key: cord-0332958-bufigko1
authors: Ocañas, Sarah R.; Ansere, Victor A.; Tooley, Kyla B.; Hadad, Niran; Chucair-Elliott, Ana J.; Stanford, David R.; Rice, Shannon; Wronowski, Benjamin; Hoffman, Jessica M.; Austad, Steven N.; Stout, Michael B.; Freeman, Willard M.
title: Differential regulation of mouse hippocampal gene expression sex differences by chromosomal content and gonadal sex
date: 2021-09-02
journal: bioRxiv
DOI: 10.1101/2021.09.01.458115
sha: cec1b7c108e03b7ff5a7643d60c12e103eaca35e
doc_id: 332958
cord_uid: bufigko1

Sex differences in the brain as they relate to health and disease are often overlooked in experimental models. Many neurological disorders, like Alzheimer’s disease (AD), multiple sclerosis (MS), and autism, differ in prevalence between males and females. Sex differences originate either from differential gene expression on sex chromosomes or from hormonal differences, either directly or indirectly. To disentangle the relative contributions of genetic sex (XX v. XY) and gonadal sex (ovaries v. testes) to the regulation of hippocampal sex effects, we use the “sex-reversal” Four Core Genotype (FCG) mouse model which uncouples sex chromosome complement from gonadal sex. Transcriptomic and epigenomic analyses of hippocampal RNA and DNA from ∼12 month old FCG mice, reveals differential regulatory effects of sex chromosome content and gonadal sex on X- versus autosome-encoded gene expression and DNA modification patterns. Gene expression and DNA methylation patterns on the X chromosome were driven primarily by sex chromosome content, not gonadal sex. The majority of DNA methylation changes involved hypermethylation in the XX genotypes (as compared to XY) in the CpG context, with the largest differences in CpG islands, promoters, and CTCF binding sites. Autosomal gene expression and DNA modifications demonstrated regulation by sex chromosome complement and gonadal sex. These data demonstrate the importance of sex chromosomes themselves, independent of hormonal status, in regulating hippocampal sex effects. Future studies will need to further interrogate specific CNS cell types, identify the mechanisms by which sex chromosome regulate autosomes, and differentiate organizational from activational hormonal effects.

Erythroid differentiation regulator 1 (Erdr1) is part of X and Y chromosome pseudoautosomal 257 region (PAR) that is able to crossover and recombine during meiosis. As such, genes within the 258 PAR have the same sequence on the X and Y chromosome and the chromosomal origin (X or Y) 259 of these transcripts cannot be determined with traditional RNA-Seq. In FCG hippocampi, Erdr1 is 260 differentially expressed by sex chromosome complement (XX v. XY), with higher levels in XX 261 animals ( Figure 2I) . 262

The eight common previously identified X/Y-chromosome sex differences (Figure 2A , In females, X-chromosome inactivation (XCI) occurs through multi-layer epigenetic mechanisms 283 that ultimately compact the inactive X-chromosome (Xi) into a heterochromatic Barr Body. Early 284 in development, the long-noncoding RNA (lncRNA) Xist is expressed from Xi and provides a cis-285 coating that recruits protein complexes, leading to changes in chromatin accessibility and DNA 286 modifications 13 . Changes in histone modifications 52 and DNA methylation stabilizes Xi in the 287 inactive state 63 . In this study, we assess the efficacy of Xi epigenetic silencing in the FCG mouse 288 model by analyzing the X chromosome DNA methylation patterning in FCG hippocampal DNA. 289

To assay DNA methylation, DNA isolated from FCG hippocampi (n=3/group) was oxidized and 290 bisulfite-converted prior to constructing whole genome libraries for sequencing on Illumina's 291 NovaSeq6000 platform. After aligning and calling methylation values, the whole genome 292 methylation levels in both CG and non-CG (CH) contexts were calculated. There were no 293 observed differences in overall whole genome methylation in CG context (mCG) (Figure 3A) by 294

When focused on the X chromosome, XX genotypes have higher mCG levels than XY genotypes, 296 regardless of sex (M v. F) (Figure 3B ; Two-way ANOVA, main effect sex chromosome 297 complement (XX v. XY), ***p<0.001). There was no difference in mCG percentages in repetitive 298 elements on the X-chromosome ( Figure 3C ; Two-way ANOVA) but in non-repetitive elements, 299 higher methylation in XX genotypes than XY genotypes regardless of their gonadal sex was 300 observed ( Figure 3D ; Two-way ANOVA, main effect sex chromosome complement (XX v. XY), 301 ***p<0.001). In non-CpG (CH) context, there was no overall difference in methylation ( Figure 3E ). 302

However, there was higher X-chromosome mCH in XY than XX, regardless of gonadal sex 303 (Figure3F; Two-way ANOVA, main effect sex chromosome complement (XX v. XY), ***p<0.001). 304

Higher XY mCH was seen in both repetitive ( Figure 2G ) and non-repetitive ( Figure 3H) elements 305 of the X-chromosome, as compared to XX (Two-way ANOVA, main effect sex chromosome 306 complement (XX v. XY), ***p<0.001). These X-chromosomal methylations trends are consistent 307 with previous reports 64 . 308

Since the sex chromosome complement (XX v. XY) difference in mCG appears to be 309 concentrated in non-repetitive elements of the X chromosome, we assessed the mCG patterning 310 in and around CpG islands, gene bodies, and CTCF binding sites. CpG islands (CGIs) are relatively long stretches (500-2000nt) of GC-rich DNA that are predominantly unmethylated 65 . 312

CGIs have higher methylation on the Xi 66 . In the FCG hippocampi, XX genotypes have higher 313 mCG within CGIs, shores, and shelves than XY genotypes, regardless of gonadal sex. The largest 314 average mCG difference occurs in the CGI followed by the shores and then shelves ( Figure 3I ; 315 Two-way ANOVA, main effect chromosome (XX v. XY)). While on average most CGIs are 316 hypermethylated in XX (over XY) genotypes, there is a small subset of genes which show 317 hypomethylation of CGIs in XX (vs. XY). 318

Similarly, gene bodies and the regions 4 kilobases upstream from the transcription start site (TSS) 319 and 4 kb downstream of the transcription end site (TES) had higher mCG in XX than in XY 320 genotypes ( Figure 3J ; Two-way ANOVA, main effect chromosome (XX v. XY)). The largest 321 difference in mCG was in the regions 4 kilobases upstream from the transcription start site (TSS), 322

inclusive of the gene promoter. 323 CCCTC-binding factor (CTCF) is a zinc-finger protein that mediates chromatin insulation and 324 gene expression by binding 12-to 20-bp DNA motifs (CTCF binding sites) and altering the 3-325 dimensional chromatin structure. CTCF has high affinity for certain RNA transcripts, including Xist 326 and anti-sense transcript Tsix, 67 which may help to differentially package the inactive and active 327 X chromatin. In the FCG hippocampus, there is lower mCG in X Chromosome CTCF binding sites 328 as compared to mCG on the whole X chromosome in all genotypes (XXF, XXM, XYF, XYM). 329

There is higher mCG in XX hippocampi than XY, regardless of gonadal sex ( Figure 3K) . The 330 magnitude of difference between XX and XY mCG in CTCF binding sites (~10%) is much greater 331 than the average difference seen across the X chromosome (~3%). 332

Together, the methylation analysis of the X chromosome in FCG hippocampi suggests that X 333 chromosome methylation is regulated by sex chromosome complement (XX v. XY) and likely not 334 influenced by gonadal status. 335

After exploring the overall levels and patterning of DNA methylation on the X chromosome, mCG 338

DMRs were called using 1 kb non-overlapping windows with minimum average difference of 10% 339 between at least two groups (Chisq-test, sliding linear model (SLIM) q<0.05) and post-hoc 340

Bonferroni corrected t-tests (XXF v. XYF, XXM v. XYM, XXF v. XXM, XYF v XYM) were used to 341 assess pairwise differences. Using these criteria, we identified 1011 DMRs between XXF and 342 XYF and 1367 DMRs between XXM and XYM, with 369 common DMRs ( Figure 4A ) on the X 343 chromosome. The 369 common DMRs appear to be evenly distributed across the X chromosome 344 with some gaps containing no DMRs ( Figure 4B ). In agreement with the average difference in 345 mCG (XX-XY) across the X chromosome, there are more hypermethylated DMRs in the XX 346 genotypes as compared to the XY genotypes ( Figure 4C ). When the DMR methylation was higher 347 in the XX genotypes, the XY genotypes had methylation values close to zero ( Figure 4D ). In 348 cases where the XX genotypes had lower DMR methylation than the XY genotypes, the XY 349 genotypes had methylation values close to 100% ( Figure 4D ). Sex-chromosomally driven DMRs Our WGoxBS data had 2-6X genome-wide coverage, which is sufficient to analyze methylation 365 values in windows and collapse certain genomic regions. In order to assess base-specific 366 methylation patterning, we performed targeted bisulfite amplicon sequencing (BSAS) within the 367 promoter region of X-chromosome genes. 368 X-linked DEAD-box RNA helicase DDX3 (Ddx3x) plays an integral role in transcription and 369 translation, as well as splicing and RNA transport. Mutations in Ddx3x have been associated with 370 intellectual disability and developmental delays 46 . In our study, Ddx3x is more highly expressed 371 in XX genotypes (XXF/XXM) as compared to XY genotypes (XYF/XYM). We amplified a region 372 of the Ddx3x promoter, containing 4 CpG sites. The average mCG across that region was higher 373

in XX genotypes as compared to XY genotypes, regardless of gonadal sex ( Figure 5A ). When 374 examining the site-specific mCG across the amplified region of the Ddx3x promoter, all 4 mCG sites have higher mCG in XX genotypes than XY genotypes ( Figure 5B ). The site-specific 376 methylation appears to be strongly regulated by sex chromosome complement, as evidenced by 377 the consistent topography of mCG. 378

Many of the consistently detected sexual dimorphisms on the X-chromosome are involved in 379 maintenance of X-chromosome inactivation through cis-coating of the Xist transcript and the 380 histone demethylase activities of Kdm6a, Kdm5d, and Uty. As such, both the X-chromosome gene 381 expression and DNA methylation appear to be strongly regulated by sex chromosome 382 complement. Although there were 2 genes (Ace2, Aff1) differentially expressed by sex, we did 383 not find any examples of X-chromosome genomic features differentially methylated by gonadal 384

Following sex-chromosomally driven sex determination, development of the gonads and 386 production of sex hormones further drives dichotomization of sexual phenotypes. In gonadal 387 males, testes produce testosterone, an androgenic hormone 68 . Androgen receptor (Ar) is a 388 hormone nuclear receptor and transcription factor that has many biological functions, including 389

proper development of male reproductive organs and secondary sex characteristics 69 . Androgens 390 have been found to effect hippocampal structure and function, as well as playing a role in 391 hippocampal-dependent behavior, long-term potentiation, and dendritic arborization 70 . 392

Ar is an X-chromosomally encoded gene and subject to X-chromosome inactivation. Although Ar 393 was not differentially expressed by sex in outside studies (Supplemental table 1 (Union)) or in 394 the present study (neither by sex (M v. F) or sex-chromosome complement (XX v. XY)), we 395 wanted to determine if gonadal sex had any effect on Ar promoter methylation. BSAS analysis of 396 22 CpGs in the Ar promoter region, showed very low (~0%) mCG in XY genotypes with close to 397 40% average methylation in XX genotypes, regardless of gonadal sex ( Figure 5C ). Each CG-site 398 within the amplified region of the Ar promoter has lower mCG (~0%) in XY genotypes compared 399 to XX genotypes (~10-60%), regardless of gonadal sex. The patterning of Ar promoter mCG is 400 well-conserved between XXF and XXM, suggesting tight regulation of Ar promoter methylation by 401 sex chromosome complement, with no effect of gonadal sex ( Figure 5D) . 402

Toll-like receptor (Tlr7) is an X-encoded pattern recognition receptor (PRR), critical in innate 403 immunity. Tlr7 recognizes single-stranded viral RNA (ssRNA) 71 and is primarily expressed on 404 microglia in the brain 72 . In response to ssRNA, Tlr7 initiates a Type I interferon (IFN) response. 405

Tlr7 was differentially expressed in outside studies (Supplemental table 1 (Union)) and by sex-406 chromosome complement in the present study ( Figure 2E ) with higher expression in XY (v. XX) 407

genotypes. The average mCG in an amplified region of the Tlr7 promoter containing 4 CG sites 408 is higher in XX genotypes over XY genotypes ( Figure 5E) . Three of the four CG sites within the 409 amplified region were higher in XX genotypes as compared to the XY genotypes, with no 410 differences by gonadal sex (Figure 5F) . 411

Xist was one X-encoded gene that was differentially expressed by sex in all outside studies 412 examined (Supplemental table 1 (Intersect)) and by sex chromosome complement in the 413 present study (Figure 1B,1E) . As a critical regulator of X-inactivation and X-chromosome dosage 414 compensation, we analyzed mCG in an amplified region of the Xist promoter in FCG hippocampi. 415

The average mCG within the amplified region of the Xist promoter was higher in XY genotypes 416 than XX genotypes, regardless of their gonadal sex ( Figure 5G) . The base-specific topography 417 of CG methylation is well conserved by sex chromosome complement (XX/XY), with no effect of 418 gonadal sex ( Figure 5H) . 419

Angiotensin-converting enzyme 2 (Ace2) is surface receptor responsible for negative regulation 420 of the renin-angiotensin system to modulate blood pressure and fluid/electrolyte balance. Ace2 421 recently gained attention as the entry receptor for the novel SARS-coronavirus 2 (SARS-CoV-422 2) 73 . Ace2 was differentially expressed by sex in the outside studies we examined (Supplemental 423 table 1 (Union)) and by gonadal sex (M v. F) in the present study ( Figure 2G) . We assessed the 424 mCG at a single CpG site within the Ace2 promoter and found higher mCG in XY genotypes as 425 compared to XX genotypes, irrespective of their gonadal sex ( Figure 5I) . 426

In summary, targeted methylation analysis confirmed that X-chromosome methylation is tightly 427 regulated, in a base-specific fashion, by sex chromosome complement, and not gonadal sex. 428

After establishing that the sex chromosome transcriptome and methylome of FCG hippocampi 430 are primarily controlled by sex chromosome complement (XX v. XY), we examined autosomal 431 regulation of sex differences. We first intersected the previous hippocampal transcriptomic studies 432 to determine steady-state sex differences in the mouse hippocampus ( Figure 6A) . Although there 433 were no sex differences in common between all studies (Supplemental Table 2 (Intersection)), 434 there were 2896 sex differences identified in at least one study (Supplemental Table 2 

We next ran GO Biological Process Over-Representation Analysis (ORA) using WEB-based 436

GEne SeT AnaLysis Toolkit (WebGestalt, www.webgestalt.org) on autosomal genes that were 437 differentially expressed by sex in at least one outside study (Supplemental Table 2 

Enriched GO pathways were visualized on a reduced directed acyclic graph (DAG) (Figure 6B ).

We identified 10 major GO terms, including cell-cell adhesion via plasma-membrane adhesion 440 molecules, connective tissue development, epithelial cell proliferation, reproductive system 441 development, urogenital system development, muscle system process, embryonic organ 442 development, gliogenesis, multicellular organismal homeostasis, and pattern specification 443 process (Supplemental Table 3 ). 444

We assessed autosomal sex differences in FCG hippocampus by sex chromosome complement three pathways (response to protozoan, response to interferon beta, and response to virus) 460 ( Figure 6F) . 461

Since we identified response to viruses and interferon-beta as pathways enriched in sex 462 differences in the FCG hippocampus, we next further examined interferon-associated genes IRF-463 7 and IFIT-3. Interferon (IFN) is part of the innate immune system important in antiviral immunity. 464

Upon viral recognition, production of IFN triggers the expression of IFN-stimulated genes (ISGs). 465 IFN-beta is a type I IFN that is activated through PRRs 74 , like Tlr7. In the brain, IFN-beta is 466 primarily expressed by microglia 72 . In mouse models of AD, IFN was found to activate microglia 467 leading to neuroinflammation and synaptic degradation. Blocking IFN signaling decreased 468 microglia activation and concomitant synapse loss. Activation of IFN pathway was also observed 469 in human AD 75 . 470

Transcription factor IRF-7 is considered a "master regulator" in type-I IFN responses 76 . Irf7 was 471 differentially expressed by sex in one of the previously examined studies (Supplemental Table 2 (Union)). Irf7 was also differentially expressed in our study by sex chromosome complement 473 (XX v. XY) and gonadal sex (M v. F) in FCG hippocampus as evidenced by RNA-Seq 474 (Supplemental Table 3 ) and RT-qPCR confirmation (Figure 6G) . Interferon-induced protein with 475 tetratricopeptide repeats 3 (IFIT3) is an antiviral RNA-binding protein which acts an intermediary 476 in the activation of IRF-3 and upregulation of IFN-beta 77 . Ifit3 was differentially expressed in one 477 of the examined outside studies (Supplemental Table 2 (Union) ). Ifit3 was also differentially 478 expressed by sex chromosome complement (XX v. XY) and gonadal sex (M v. F) in FCG 479 hippocampus as evidenced by RNA-Seq (Supplemental Table 3 ) and RT-qPCR confirmation 480 ( Figure 6H) . 481

Antigen processing and presentation was another pathway that was over-represented in our 

After analyzing autosomal sex differences in the FCG hippocampus, we assessed autosomal 492 methylation in CG and CH context by WGoxBS. Overall, there were no differences in autosomal 493 mCG ( Figure 7A) , with no difference in autosomal mCG in repetitive ( Figure 7B ) or non-repetitive 494 ( Figure 7C ) elements. There also was no difference in average autosomal mCH (Figure 7D) , 495 with no difference in autosomal mCH in repetitive (Figure 7E ) or non-repetitive ( Figure 7F ). There 496 also were no apparent differences in autosomal mCG patterning across CGI, shores, and shelves 497 ( Figure 7G) , gene bodies/flanking regions (Figure 7H) , or CTCF-binding sites/flanking regions 498 ( Figure 7I) . 499

After exploring the overall levels and patterning of DNA methylation on the autosomes, we called 502 mCG DMRs using 1 kb non-overlapping windows with minimum average difference of 10% 503 between at least two groups (Chisq-test, sliding linear model (SLIM) q<0.05) and post-hoc Bonferroni corrected t-tests (XXF v. XYF, XXM v. XYM, XXF v. XXM, XYF v XYM, p<α=0.0125) 505 were used to assess pairwise differences. Using these criteria, we identified 2363 DMRs between 506 XXF and XYF and 3031 DMRs between XXM and XYM, with 45 common sex chromosomally-507 regulated autosomal DMRs (Figure 8A) . Hierarchical clustering of the DMRs showed three 508 distinct clusters, with a set of 19 DMRs with higher mCG in XY genotypes (XY >XX), 25 DMRs 509 with higher mCG in XX genotypes (XX>XY), and 1 discordant DMR (Figure 8B) . DMRs differentially regulated by both sex chromosome and gonadal sex were over-represented 524 in pathways involved in synaptic signaling, cell motility, and neurogenesis ( Figure 8G) . Thus, 525 despite a strong immune-related transcriptomic signature, differential methylation appears to be 526 mostly involved in neuron-related pathways. We believe that this is due to the relatively low 527 percentage of microglia within the brain and warrants further cell-type specific studies focusing 528 on microglia. 529

The study of sex effects in brain health and disease have begun receiving the needed 531 experimental attention in neuroscience studies. Not only do the sexual dimorphisms, differences, 532 and divergences 81 need to be characterized but also the regulatory mechanisms giving rise to 533 these sex effects. While hormonal mechanisms (both organizational and activational) have been 534 the most studied, the potential regulation of sex effects by sex chromosomes, either 535 independently, and in concert with hormones, has received relatively limited attention. Recent 536 reports however, support the idea that sex chromosomal content is a central regulatory factor 82, 83 . 537

To begin characterizing the effects of sex chromosome content on epigenetic regulation of sex 538 chromosomes and autosomes, this study used the four core genotype (FCG) model 33 to examine 539 hippocampal DNA modifications and gene expression by gonadal sex (M vs F) and sex 540 chromosome content (XX vs XY) in roughly one-year old mice. 541

We first localized the translocation of Sry to an intergenic region of chromosome 3. This agrees 542 with prior imaging studies 41 and provides a precise location of the insertion. Importantly we also 543 found no evidence of other translocation sites in the genome. While there are ~13 copies of Sry 544

insertions of Sry they are not within an annotated gene. This could raise concerns about ectopic 545

Sry expression, but no evidence of Sry expression in the hippocampus was found, as would be 546 expected, providing evidence that normal tissue-specific regulation is occurring. 547

After performing paired transcriptomic and DNA methylation sequencing, a number of principle 548 findings were evident. For gene expression regulation, sex dimorphisms and differences in the 549 gene expression of X and Y encoded genes are principally driven by sex chromosome content 550

and not gonadal sex. Autosomally encoded gene expression differences are regulated by both 551 sex chromosomes and gonadal sex. While in a sense this may not be surprising, we are unaware 552 of prior data in the brain examining this point. The mechanism for this differential regulation may 553 lie, in part, in the X chromosome encoded histone demethylase Kdm6a and Y-encoded Uty and 554

Kdm5d as DEGs were enriched by H3-K27 and H3-K4 demethylation responsiveness. Future 555 work will need to manipulate individual X-and Y-encoded sexually dimorphic genes in the context 556 of the FCG model to determine the relative contributions of these to the autoregulation of the sex 557

Analyses of hippocampal DNA methylation patterns across the XXF, XXM, XYM, and XYF 559 genome revealed a similar differential effect on autosomes and sex chromosomes. While whole 560 genome mCG levels did not vary by sex chromosome content or gonadal status, X chromosome 561 mCG in non-repetitive elements were lower in XYF and XYM compared to XX animals. This lower 562 level of mCG was also enriched in CpG Islands, promoter regions, and CTCF binding sites of the 563 X chromosome and likely reflects that XX mice have one inactive X (Xi). This interpretation is 564 bolstered by analysis of differentially methylated regions which are principally higher in XX vs XY 565 and correlate to mRNA expression of X encoded genes. Importantly this potential inactivation was 566 unaffected by gonadal status. 567

Conversely non-CpG methylation (mCH) was higher in XY versus XX mice irrespective of gonadal 568 status. Higher levels of X chromosome mCH have been reported in the liver 66 and have been 569

suggested to indicate Xi escape 84 . As these analyses were performed on tissue homogenates 570 future studies will need to examine mCH in a cell type specific manner as mCH levels are much 571 higher in neurons than other CNS cell types 84 . 572

The patterns of autosomal DNA methylation by sex chromosome content and gonadal sex 573 presented a very different profile. There were no overall differences in mCG or mCH levels. 574

Rather differentially methylated regions were evident by both sex chromosome content and 575 gonadal sex. Unlike the X chromosome, differences were not found in CGI regions but were most 576 enriched in promoter flanking regions. Furthermore, differentially methylated regions were both 577 higher and lower in comparisons by sex chromosome and gonadal sex unlike in the X 578 chromosome which were almost uni-directional. 579

Taken together these findings are consistent with the hypothesis that the sex chromosomes have 580 gonadal sex independent effects on the hippocampal epigenome and transcriptome. The use of 581 the FCG mouse model allows for this demonstration for the first time in the brain. However, a 582 number of questions remain to be answered in future studies. These principally consist of further 583 controlling for gonadal hormone status but analyzing FCG mice that have be gonadectomized 584 after development ~2-3 months of age. This will control for any activation hormonal differences 585 between the genotypes. It is worth noting that these studies were conducted in adult mice ~12 586 months of age. 587

Most importantly for future studies, analysis of specific cell types is needed. The gene expression 588 and epigenomic differences between neuronal, glial, and other cell types of the CNS is well 589 described. Examining specific cell populations will increase the signal to noise from future 590 molecular studies. Further investigation is highly warranted though given the significant effects of 591 sex chromosome regulation of gene expression and DNA modification patterns in cis of the X 592 chromosome and in trans of the autosomes. and quantified by qPCR (KAPA Biosystems). Libraries were then normalized to 4 nM, pooled, 681 denatured, and diluted for sequencing on Illumina Hiseq2500 in a 2x100 bp fashion. 682

Following sequencing, reads were trimmed, aligned, differential expression statistics and 684 correlation analyses were performed in Strand NGS software package (Agilent), as previously 685 described 43 . Reads were aligned against the Mm10 build of the mouse genome (2014.11.26). 686

Alignment and filtering criteria included: adapter trimming, fixed 2bp trim from 5' and 6bp from 3' 687 ends, a maximum number of one novel splice allowed per read, a minimum of 90% identity with 688 the reference sequence, a maximum of 5% gap, trimming of 3' end with Q<30. Alignment was 689 performed directionally with Read 1 aligned in reverse and Read 2 in forward orientation. Reads 690 were filtered based on the mapping status and only those reads that aligned normally (in the 691 appropriate direction) were retained. Normalization was performed with the DESeq algorithm 91 . 692

Transcripts with an average read count value >20 in at least 100% of the samples in at least one 693 group were considered expressed at a level sufficient for quantitation per tissue. Those transcripts 694 below this level were considered not detected/not expressed and excluded, as these low levels 695 of reads are close to background and are highly variable. A fold change >|1.25| cutoff was used to eliminate those genes which were unlikely to be biologically significant and orthogonally Global levels of mCG, hmCG, and mCH were analyzed as previously described 43, 92 Before 729 aligning, paired-end reads were adaptor-trimmed and filtered using Trimmomatic 93 0.35. End-730 trimming removed leading and trailing bases with Q-score<25, cropped 4 bases from the start of 731 the read, dropped reads less than 25 bases long, and dropped reads with average Q-score<25. 732

Unpaired reads after trimming were not considered for alignment. Alignment of trimmed OxBS-733 converted sequences was carried out using Bismark 94 0.16.3 with Bowtie 2 95 against the mouse 734 reference genome (GRCm38/mm10). Bams were de-duplicated using Bismark. Methylation call 735 percentages for each CpG and non-CpG (CH) site within the genome were calculated by dividing 736 the methylated counts over the total counts for that site in the oxidative bisulfite -converted 737 libraries (OXBS). Genome-wide CpG and CH methylation levels were calculated separately. BAM 738 files generated during alignment were run through MethylKit in R 96 to generate context-specific 739 (CpG/CH) coverage text files. Bisulfite conversion efficiency for C, mC, and hmC was estimated 740 using CEGX spike-in control sequences. Untrimmed fastq files were run through CEGX QC v0.2, 741 which output a fastqc_data.txt file containing the conversion mean for C, mC, and hmC. 742

CpG text files were read into methylKit v XXXX and converted to an object. The mouse genome 744 was tiled in 200 nt non-overlapping windows. Each window was filtered for a minimum count of 745 10. Samples were then united and compared for windows covered in all samples. Differentially 746 methylated regions (DMRs) were called using default parameters. DMRs were filtered to 747 differences that were >5% different between at least two groups and had a SLIM-generated q-748 value less than 0.05. There were 13010 windows that met these criteria. The methylDiff object 749 was intersected with the methylBase object to calculate the % methylation for each window that 750 passed the described filtering. Next, 4 pairwise t-tests (XXF v XYF, XXM v XYM, XXF v XXM, 751

XYF v. XYM) were conducted and corrected for the four comparisons using BHMTC and an 752 alpha<0.05. 753 

Sex differences in hippocampal cognition and neurogenesis

Mammalian sex determination-insights from humans and mice

Organizing action of prenatally 760 administered testosterone propionate on the tissues mediating mating behavior in the female 761 guinea pig

Gonadectomy reduces the density of 763 androgen receptor-immunoreactive neurons in male rat's hippocampus: testosterone 764 replacement compensates it

Sex differences in behavioral and neurochemical effects of gonadectomy and 766 aromatase inhibition in rats

THC, and hormones: Effects on density and sensitivity of CB(1) 768 cannabinoid receptors in rats

Activational and organisational effects of 770 gonadal steroids on sex-specific acetylcholine release in the dorsal hippocampus

Androgen receptor is a negative regulator of 776 contextual fear memory in male mice

Gonadal steroids regulate number of astrocytes 778 immunostained for glial fibrillary acidic protein in mouse hippocampus

Conceptual frameworks and mouse models for studying sex differences in 781 physiology and disease: why compensation changes the game

Estrogen Replacement Therapy for Treatment of Mild to Moderate 783

Alzheimer DiseaseA Randomized Controlled Trial

X Inactivation and Escape: Epigenetic and Structural 785 Features

Turner syndrome and sexual differentiation of the brain: 787 implications for understanding male-biased neurodevelopmental disorders

Klinefelter's syndrome (47,XXY) is in excess among men with Sjogren's 790 syndrome

A general theory of sexual differentiation

X-chromosome inactivation: the molecular basis of silencing

X Inactivation and Escape: Epigenetic and Structural 796 Features

X-inactivation profile reveals extensive variability in X-linked gene 798 expression in females

Genes that escape from X inactivation

Escape from X chromosome inactivation and female 802 bias of autoimmune diseases

Escape from X Chromosome Inactivation and the 804 Female Predominance in Autoimmune Diseases

The Mystery of X Chromosome Instability in Alzheimer's Disease

CNS-wide Sexually Dimorphic Induction of the Major Histocompatibility 808 Complex 1 Pathway With Aging

Sexually divergent DNA methylation patterns with hippocampal aging

Transcriptomic analysis of the hippocampus from six inbred strains of mice 812 suggests a basis for sex-specific susceptibility and severity of neurological disorders

Sex differences in the molecular signature of the 815 developing mouse hippocampus

Sex-differential DNA methylation and associated regulation networks in human 817 brain implicated in the sex-biased risks of psychiatric disorders

Mouse models for evaluating sex chromosome effects that cause sex differences in 820 non-gonadal tissues

A model system for study of sex chromosome effects on sexually dimorphic 822 neural and behavioral traits

Sex differences in mouse cortical thickness are independent of the 824 complement of sex chromosomes

Neonatal mice possessing an Sry transgene show a masculinized pattern of 826 progesterone receptor expression in the brain independent of sex chromosome status

What does the "four core genotypes" mouse model tell us about sex 829 differences in the brain and other tissues?

Sex chromosome genes directly affect brain sexual 831 differentiation

Sex chromosome complement affects nociception in tests of acute and chronic 833 exposure to morphine in mice

Sex difference in neural tube defects in p53-null mice is caused by differences in 835 the complement of X not Y genes

A role for sex chromosome complement in the female bias in 837 autoimmune disease

Female XX sex chromosomes increase survival and extend 839 lifespan in aging mice

A second X chromosome contributes to resilience in a mouse model of 841

Alzheimer's disease

<em>Sry</em>: the master switch in mammalian sex 843 determination

Four core genotypes mouse model: localization of the Sry transgene and bioassay 845 for testicular hormone levels

Sex differences in the molecular signature of the 847 developing mouse hippocampus

Tamoxifen induction of Cre recombinase does not cause long-lasting or 849 sexually divergent responses in the CNS epigenome or transcriptome: implications for the design 850 of aging studies

Early-life DNA 852 methylation profiles are indicative of age-related transcriptome changes

Escape from X Inactivation Varies in Mouse Tissues

Mutations in DDX3X Are a Common Cause of Unexplained Intellectual 857 Disability with Gender-Specific Effects on Wnt Signaling

X-exome sequencing of 405 unresolved families identifies seven novel intellectual 859 disability genes

Demethylase-Independent Function in Mouse Embryonic Development

Xist localization and function: new insights 864 from multiple levels

KDM5D-mediated H3K4 demethylation is required for sexually dimorphic 866 gene expression in mouse embryonic fibroblasts

DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell 868 formation and transcriptional programs

High-resolution analysis of epigenetic changes associated with X inactivation

Effects of DNA methylation on DNA-binding proteins and gene expression

Methylated DNA and MeCP2 recruit histone deacetylase to repress 874 transcription

Methylated DNA-binding domain 1 and methylpurine-DNA glycosylase link 876 transcriptional repression and DNA repair in chromatin

Methyl-CpG binding domain 1 (MBD1) interacts with the Suv39h1-HP1 879 heterochromatic complex for DNA methylation-based transcriptional repression

Two highly related p66 proteins comprise a 882 new family of potent transcriptional repressors interacting with MBD2 and MBD3

DNA methylation and genomic imprinting

Methylation of the 886 hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-887 chromosome inactivation

Polymerase Chain 890 Reaction-Aided Genomic Sequencing of an X Chromosome-Linked CpG Island: Methylation 891 Patterns Suggest Clonal Inheritance, CpG Site Autonomy, and an Explanation of Activity State 892 Stability

DNA methylation of two X 894 chromosome genes in female somatic and embryonal carcinoma cells

TACC3 mediates the association of MBD2 with histone acetyltransferases 897 and relieves transcriptional repression of methylated promoters

DNA methylation profiles of human active and inactive X chromosomes

Tamoxifen induction of Cre recombinase does not cause long-lasting or 902 sexually divergent responses in the CNS epigenome or transcriptome: implications for the design 903 of aging studies

DNA methylation of intragenic CpG islands depends on their 905 transcriptional activity during differentiation and disease

Dosage compensation and DNA methylation landscape of the X chromosome 908 in mouse liver

Locus-specific targeting to the X chromosome revealed by the RNA interactome 910 of CTCF

Does Gender Leave an Epigenetic Imprint on the 912 Brain?

Androgen Receptor Structure, Function and Biology: From Bench 914 to Bedside

Androgen Modulation of Hippocampal 916 Structure and Function

Toll-like receptor signaling pathways

An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and 919 Vascular Cells of the Cerebral Cortex

The protein expression profile of ACE2 in human tissues

Type I interferon pathway in CNS homeostasis and neurological disorders

Type I interferon response drives neuroinflammation and synapse loss in 925 Alzheimer disease

IRF-7 is the master regulator of type-I interferon-dependent immune responses. 927

Antiviral Signaling by Bridging MAVS and TBK1

Neuroinflammation: 931 Microglia and T Cells Get Ready to Tango

Neuroglial expression of the MHCI pathway and PirB receptor is 933 upregulated in the hippocampus with advanced aging

CNS-wide Sexually Dimorphic Induction of the Major Histocompatibility 935 Complex 1 Pathway With Aging

Sex differences in the 937 brain: the not so inconvenient truth

A second X chromosome contributes to resilience in a mouse model of 939

Alzheimer's disease

X chromosome escapee genes are involved in ischemic sexual dimorphism through 941 epigenetic modification of inflammatory signals

Global epigenomic reconfiguration during mammalian brain development

The senescence-associated secretory 945 phenotype: the dark side of tumor suppression

Cellular hallmarks of aging emerge in the ovary prior to primordial follicle 947 depletion

Focused, high accuracy 5-methylcytosine quantitation 949 with base resolution by benchtop next-generation sequencing

Sod1) mimics an age-related increase in absolute mitochondrial DNA copy number in 953 the skeletal muscle

Detecting DNA cytosine methylation using nanopore sequencing

Hippocampal subregions exhibit both distinct and shared transcriptomic 957 responses to aging and nonneurodegenerative cognitive decline

Differential expression analysis for sequence count data

Inducible cell-specific mouse models for paired epigenetic and 962 transcriptomic studies of microglia and astroglia

Trimmomatic: a flexible trimmer for Illumina sequence 964 data

Bismark: a flexible aligner and methylation caller for Bisulfite-Seq 966 applications

Fast gapped-read alignment with Bowtie 2

methylKit: a comprehensive R package for the analysis of genome-wide DNA 970 methylation profiles

Author contributions

Ocañas: first author, design of the study, execution of experiments, data acquisition, 974 analysis, and interpretation, figure generation

Ansere: execution of experiments, data acquisition, analysis, and interpretation, figure 976 generation

Tooley: execution of experiments, data acquisition, analysis, and interpretation, figure 978 generation

Niran Hadad: data analysis and interpretation, figure generation

Chucair-Elliott: design of the study, data interpretation

Stanford: design of the study, data analysis and interpretation

Shannon Rice: execution of experiments, data acquisition, analysis, and interpretation 983

Benjamin Wronowski: execution of experiments, data acquisition, analysis, and interpretation, 984 figure generation

Hoffman: design of the study, execution of experiments, data analysis and 986 interpretation

Austad: design of the study, data analysis and interpretation, manuscript writing and 988

Stout: design of the study, execution of experiments, data acquisition, analysis, and 990 interpretation

Corresponding author, design of the study, data analysis and 992 interpretation, figure generation

Transcriptomic analysis of sex chromosomal driven differential expression of sex 1017 chromosome encoded genes in adult FCG hippocampi. DNA and RNA were isolated from FCG 1018 hippocampi (n=10-16/group). mRNA expression was assessed by RT-qPCR

Results were compared to previously published hippocampal transcriptomic sex 1020 differences. Boxplots represent median, interquartile range, and minimum/maximum normalized gene 1021 expression. A) Comparison of four previous hippocampal transcriptomic studies, shows 168 sex 1022 chromosome-encoded sex differences in wild-type mice across studies with eight genes common between 1023 all studies (Xist Ddx3x, Kdm6a, Eif2s3x, Kdm5d, Eif2s3y, Uty, Ddx3y) B) In the FCG hippocampus, RT-1024 qPCR of X-chromosome encoded gene

Two-way ANOVA, main effect of sex-chromosome complement

C-D) RT-qPCR of Y-chromosome encoded genes shows similar levels of expression of (C)

Two-way ANOVA, 1028 main effect of sex-chromosome complement (XX v. XY), ***p<0.001). E) RNA-Seq analysis of X-1029 chromosome encoded genes showed 20 genes that are differentially expressed by sex chromosome (XX 1030 vs. XY) but not by sex (M v. F). F-G) RNA-seq analysis of X chromosome genes revealed only two genes 1031 (Aff2, Ace2) differentially expressed by sex (M v. F) and not by sex chromosome complement

Ace2 had higher expression in males than females regardless of their sex chromosome

H-I) RNA-Seq analysis of Y chromosome encoded 1034 genes identified 5 differentially expressed genes by sex chromosome (XX vs. XY) but not by sex

There were no Y chromosome genes that were differentially expressed by sex (M v. F). H) Four of the 1036 genes (Kdm5d, Eif2s3y, Ddx3y, Uty) show no expression in XX genotypes. I) Located in the pseudo 1037 autosomal region (PAR) of the X/Y-chromsomes, Erdr1 shows higher expression in XX genotype

Comparing the union of previous hippocampal studies described in (A) to the FCG sex chromosome genes 1039 differentially expressed between XX and XY genotypes, yields 18 common genes. K) GO Ontology analysis 1040 of the 18 genes from (J), identified four significantly enriched biological pathways 1041

Figure 3. X chromosome levels of methylation in FCG hippocampus by WGoxBS. DNA was isolated from 1044 FCG hippocampi (n=3/group). Methylation in CpG (CG) and non-CpG (CH) contexts was assessed by 1045

The differences in X-chromosome mCH are seen in both (E) repetitive and (F) non-repetitive elements 1053 of the genome. mCG levels were calculated with respect to genic regions by binning 200 nucleotides in 1054 flanking regions and region-size dependent bins within the genic region (CGI, gene body, and CTCF) as to 1055 maintain the same number of bins for each feature. The average for all (I) CGI, (J) Gene Body, and (C) CTCF 1056 were assessed for each of the FCG (XXF, XXM, XYF, XYM) and plotted as averages with 95% CI. I) X-1057 chromosome CpG Islands (CGI), shores, and shelves have higher levels of mCG in XX genotypes as 1058 compared to XY. The greatest difference in mCG is in the CGI. J) X-chromosome gene bodies and flanking 1059 regions (+/-4 Kb) have higher levels of mCG in XX genotypes as compared to XY. The greatest difference 1060 in mCG is upstream of TSS (ie. promoter region). K) X-chromosome CTCF binding sites have higher levels 1061 of

26 genes had main effects of chromosome and sex. D) Principal component analysis of 1117 differentially expressed autosomal genes showed separation of sex (M v. F) in the first component (33.9%) 1118 and separation of the chromosome (XX v. XY) in the second component (20.2%). E) Hierarchical clustering 1119 of differentially expressed autosomal genes shows separation of genotypes by sex and sex chromosome 1120 complement. F) ORA of the autosomal sex differences identified in the FCG hippocampus revealed 4 1121 pathways (Supplemental Table 3) differentially regulated by sex chromosome complement and/or 1122 gonadal sex. G-L) Differential expression of select genes was

main effect of sex chromosome complement (XX v. XY) or gonadal sex (M v. F), *p<0

01, and ***p<0.001)

Tecan Genomics, Inc., Redwood City, CA) as previously described 43,92 . Briefly, 1 µg of gDNA in 709 50 µl 1X low-EDTA TE buffer was sheared with a Covaris E220 sonicator (Covaris, Inc., Woburn, 710 MA) to an average of 200 base pairs. Sheared products were sized by capillary electrophoresis 711 (DNA HSD1000, Agilent) and cleaned using an Agencourt bead-based purification protocol. After and RNA were isolated from FCG hippocampi (n=10-16/group). mRNA expression was assessed by 1107 stranded RNA-Seq (n=5-6/group) and RT-qPCR (n=10-16/group). Results were compared to previously 1108 published hippocampal transcriptomic sex differences. Boxplots represent median, interquartile range, 1109and minimum/maximum normalized RQ. A) Comparison of four previous hippocampal transcriptomic 1110 studies (Supplemental Table 2 ), shows 2896 autosomal-encoded sex differences in wild-type mice across 1111all studies with no genes in common between all studies. B) GO Biological Process Over-Representation 1112Analysis (ORA) of the autosomal sex differences identified in previous studies revealed 10 major pathways 1113 differentially regulated by sex in the mouse hippocampus (Supplemental Table 3