Male mice with large inversions or deletions of X-palindrome arms are fertile and express their associated genes post-meiosis

Large (>10 kb) palindromic sequences are enriched on mammalian sex chromosomes. In mice, these palindromes harbor gene families (≥2 gene copies) expressed exclusively in post-meiotic testicular germ cells, at a time when most single-copy sex-linked genes are transcriptionally repressed. This distinct expression pattern led to the hypothesis that containment within palindrome structures or having ≥2 gene enables post-meiotic gene expression. We tested these two hypotheses by using CRISPR to precisely engineer large (10’s of kb) inversions and deletions of X chromosome palindrome arms for two regions carrying the mouse 4930567H17Rik and Mageb5 gene families. We found that 4930567H17Rik and Mageb5 gene expression is unaffected in mice carrying palindrome arm inversions, suggesting that palindromic structure is not important for mediating palindrome-associated gene expression. We also found that 4930567H17Rik and Mageb5 gene expression is reduced by half in mice carrying palindrome arm deletions, allowing us to test whether palindrome-associated genes are sensitive to reduced expression levels resulting in spermatogenic defects. Male mice carrying palindrome arm deletions of 4930567H17Rik or Mageb5, however, are fertile, have normal testis histology, and show no aberrations in spermatogenic cell population frequencies via FACS quantification. Together, these findings suggest that large palindromic structures on the sex chromosomes are not necessary for their associated genes to evade post-meiotic transcriptional repression and that these genes are not sensitive to reduced expression levels. Large sex chromosome palindromes may thus be important for other reasons, such as the long-term evolutionary stability of their associated gene families.


Introduction:
In humans and mice, the sex chromosomes are enriched for large (>10kb), nearly identical (>99% nucleotide identity) segmental duplications in palindromic orientation [1][2][3][4] . In mice, genes harbored within large X chromosome palindromes are expressed predominantly or exclusively in post-meiotic testicular germ cells 3 . This specific expression pattern is surprising, because most single-copy X-linked genes are transcriptionally silenced post-meiosis [5][6][7][8] . The mechanism by which palindrome-associated genes escape transcriptional repression is unknown; however, two hypotheses have been suggested to explain this distinct expression pattern. First, palindromes may form secondary structures (e.g. palindrome arms pairing to form a hairpin) enabling their associated genes to evade transcriptional repression 3 . Intrachromosomal synapsis of palindrome arm pairing could facilitate the evasion of post-meiotic gene repression, which itself is a consequence of asynapsis-triggered meiotic sex chromosome inactivation [9][10][11] . Second, X-palindromic genes may be sensitive to reduced expression levels and thus require ≥2 gene copies 3 . Consistent with this, the mouse X chromosome carries other non-palindromic genes that are in multiple copies and are expressed specifically in post-meiotic cells 3 . To test the two hypotheses, individual palindrome arms must be genetically manipulated, in vivo.
To rigorously test whether palindrome structure or gene copy number is important for postmeiotic expression, we genetically dissected two mouse X-palindromes. We utilized CRISPR to generate large-scale (10's of kb) inversions and deletions in mice of two X-palindrome arms harboring the 4930567H17Rik and Mageb5 (Melanoma antigen gene family member b5) gene families. We chose these two X-palindromes because of their canonical features of palindromes across mammals; they have >99% percent nucleotide identity between the two arms, the arms are >10kb, and they harbor a gene family expressed specifically in post-meiotic testicular germ cells. We also selected these two gene families because they have sequence family variants between the two gene copies, enabling detection of palindrome arm-specific expression. We found that for the 4930567H17Rik and Mageb5 palindromic gene families, palindrome structure is not necessary for regulating their associated post-meiotic gene expression. We observed that deletion of a single palindrome arm, for both the 4930567H17Rik and Mageb5 gene families, reduces gene expression levels by half; however, reduced expression levels did not lead to male infertility or spermatogenic defects in either case. This suggests that palindromes enrichment on the sex chromosomes is important for other reasons and that there are alternative, unknown mechanisms for palindrome-associated genes to evade post-meiotic repression.

Generation of 4930567H17Rik and Mageb5 palindrome arm inversions and deletions
To generate mice carrying palindrome arm inversions and deletions we used CRISPR with dual sgRNAs (single guide RNAs). sgRNAs were designed to unique sequences flanking the targeted palindrome arm, as close to the edge of the arm as possible. Since sgRNA cutting efficiency varies between sgRNAs, we tested their activity via pronuclear injection of an sgRNA/Cas9 expression plasmid, pSpCas9(BB)-2A-GFP (pX458) 12 in mouse zygotes. The surviving mouse zygotes were allowed to develop into 64-128 cell blastocysts and the cutting efficiency of each sgRNA was determined by PCR of the cut sties and Sanger sequencing of purified PCR products to identify local edits at the target sites.
Once active sgRNAs were identified for both sides of the targeted palindrome arm, two pX458M plasmids encoding the sgRNAs and Cas9 together with a single-stranded oligonucleotide were pronuclear injected into hybrid C57BL/6JxSJL hybrid mouse zygotes. We added single-stranded oligonucleotides with sequence homology flanking the junction boundaries, to promote either inversions or deletions. The zygotes were generated from mating C57BL/6J females to C57BL/6JxSJL males so that all targeted X chromosomes were C57BL/6J. Blastocysts from the pronuclear injection where implanted into pseudopregnant females. Genomic DNA from resulting pups was screened via PCR and Sanger sequencing for the inversion and deletion junctions. At least two independent mouse lines were obtained for inversions and deletions of the 4930567H17Rik and Mageb5 palindrome arms. Male and female mice were able to germline transmit both the 4930567H17Rik and Mageb5 deletions and inversions, thus their overall health was unaffected by our CRISPR-mediated chromosome engineering.

Mice and testis sample collection
All mice carrying an inversion or deletion of a single palindrome arm were backcrossed to C57BL/6J (N2-N7). Backcrossing was performed to minimize any possible CRISPR-mediated off-target effects. For assessing fecundity (average litter size per male), we mated mutant males to CD1 females. Testes were collected from 2-6 month old males for all experiments. We used wild-type littermate controls whenever possible and if not available, we used age-matched controls from the same breeding line. The alleles for the first mouse lines of each type were

Preparation of adult testis cDNA for qPCR
Intron-spanning primers, when possible, were used to perform qPCR on adult testis cDNA preparations. One testis per mouse was used to isolate RNA using Trizol (Invitrogen) following the manufacturers recommendations. 5 μg of total RNA was DNase treated with TurboDNase (Ambion) and reversed transcribed using Superscript II (Invitrogen) and oligonucleotide (dT) primers following the manufacturers protocol. qPCR was performed in triplicate using Power SYBR Green master mix (Thermo Fisher Scientific) on a 7500 Real-time PCR thermalcycler (Applied Biosystems). Trim42 (Tripartite motif-containing 42) was used as a normalization control, because it is expressed specifically in the same post-meiotic testicular cells and at similar levels as 4930567H17Rik and Mageb5. The Delta-delta Ct method, with Trim42 as the normalization control was used determine gene expression differences.

Testis Histology
Testes were fixed overnight in Bouin's solution, paraffin embedded, sectioned to 5 μm, and stained with Periodic acid Schiff (PAS) and hematoxylin. Sections were visualized under a light microscope and specific germ cell populations were identified by their location within a tubule, nuclear size, and chromatin pattern 13 .

FACs-based estimates of round spermatid frequencies
We largely followed a previously published protocol 14 to isolate round spermatids (1n) and spermatocytes (4n). Briefly, we disassociated cells from a single testis by enzymatic treatment with Collagenase type I, DNase I (Worthington Biochemical Corporation), and Trypsin (Life Technologies). The cell suspension was passed through cell strainers (100 µm and 40 µm) and incubated with Hoechst 33342 (Life Technologies) for DNA content and propidium iodide (Acros Organics) for cell viability. Cell sorting was performed on a FACSAria II cell sorter (BD Biosciences). The purity of each sort was determined via fluorescence microscopy visual inspection of 100 cells morphology and nuclear staining with DAPI.

RNA-seq analyses
RNA-seq analyses were conducted by analyzing previously published datasets. Specifically, mouse tissue panel data was analyzed from GSE41637 15 , ovary data from GSE43520 16 and sorted testicular germ cell populations from GSE49624 17 . Alignments were performed using Tophat 18 with the mm10 mouse reference genome, a refFlat file with RefSeq gene annotations and --max-multihits set to 240; otherwise standard default parameters were used. We used Cufflinks 18 using the refFlat gene annotation file to estimate expression levels as fragments per kilobase per millions of mapped fragments (FPKM).

Dot plots
Self-symmetry triangular dot plots that show repeats within a sequenced region were generated from a custom Perl script that can be found at http://pagelab.wi.mit.edu/material-request.html.

Results:
The mouse X chromosome harbors eight singleton palindromes.
Large palindromes on mammalian sex chromosomes are typically found as isolated pairs of palindrome arms (singleton palindromes) or in complex arrays of palindromes. We investigated singleton palindromes, because they are more commonly found across mammalian sex chromosomes and can be genetically manipulated in vivo more precisely. Of the eight singleton palindromes on the mouse X chromosome (Table 1 and Figure 1A), we selected two harboring the 4930567H17Rik and Mageb5, because they share canonical features of sex chromosome palindromes: >10kb, >99% nucleotide identity between palindrome arms, harbor genes expressed specifically in testicular germ cells, and have a spacer sequence between the palindrome arms (Table 1 and Figure 1B). Additionally, the palindrome carrying the 4930567H17Rik gene family has the longest palindrome arm (65kb), for a singleton palindrome, which if we can delete and invert in mice, will serve as a proof of principle for the manipulation of shorter palindrome arms.
We wanted to ensure that the 4930567H17Rik and Mageb5 gene families are expressed exclusively in post-meiotic round spermatids and that both copies are expressed. By reanalyzing previously published RNA-seq datasets, we find that both gene families are expressed exclusively in round spermatids ( Figure 1C). We consider the low levels of expression in spermatogonia to be due to contamination of round spermatids in the spermatogonial populations during sorting. To determine if both 4930567H17Rik and Mageb5 gene copies are expressed, we utilized individual nucleotide differences between gene copies.
Sequencing of RT-PCR products for both 4930567H17Rik and Mageb5 show that both gene copies are expressed ( Figure 1D). Having confirmed that both gene copies are expressed exclusively in post-meiotic round spermatids, we proceeded to delete and invert individual palindrome arms to assess the importance of palindrome structure and gene copy number. After normalization, we indeed find that 4930567H17Rik DelArm/Y and Mageb5 DelArm/Y mice, across two independent mouse lines, express their associated genes at approximately half the levels of wild-type males ( Figure 3B). The reduction of gene expression by half allows us to test whether the 4930567H17Rik and Mageb5 gene families are sensitive to reduced gene expression levels, resulting in males that are infertile with defects in post-meiotic spermatogenic development.

Male mice carrying deletions of individual palindrome arms do not exhibit defects in fecundity, testis histology, or numbers of round spermatids.
We performed a systematic characterization of fecundity and post-meiotic spermatogenic development in 4930567H17Rik DelArm/Y and Mageb5 DelArm/Y mice. We found that 4930567H17Rik DelArm/Y and Mageb5 DelArm/Y mice are fertile and produce litter sizes that are similar to wild-type controls ( Figure 4A). To detect potential defects in post-meiotic spermatid development, we examined testis histological sections of 4930567H17Rik DelArm/Y and Mageb5 DelArm/Y mice. We did not observe defects in spermatid morphology, formation of the acrosome, initiation of spermatid elongation or their formation or the presence of abnormal elongated spermatids in the epithelium. To assess whether the number of round spermatids were affected in 4930567H17Rik DelArm/Y and Mageb5 DelArm/Y mice, we quantified the number of round spermatids per testis as the ratio of round spermatids/spermatocytes (control) via FACs.
We find that the ratio of round spermatids/spermatoyctes of 4930567H17Rik DelArm/Y and Mageb5 DelArm/Y mice is similar to wild-type males ( Figure 4B), which is consistent with their testis weights also being similar. Altogether, 4930567H17Rik DelArm/Y and Mageb5 DelArm/Y mice do not exhibit detectable defects in fecundity and post-meiotic spermatid development.

Discussion
Our finding that palindrome structure is not necessary for facilitating post-meiotic gene expression for 4930567H17Rik and Mageb5 gene families leaves open the question as to why palindromes are heavily enriched on both the mammalian X and Y chromosomes. It is possible that palindromes, both on the X and Y chromosomes, are necessary for long-term evolutionary stability of the genes they harbor in order to rapidly purge deleterious mutations via gene conversion 19 .
Our findings also show that the 4930567H17Rik and Mageb5 gene families are not sensitive to reduced gene expression levels, when a single palindrome arm deletion reduces the gene copy number from 2 to 1. This, together with our findings on palindrome structures, suggests there are alternative mechanisms for X-palindromic genes to be expressed on the otherwise transcriptionally repressed X chromosome. There are a small number of X-linked single-copy genes expressed in round spermatids 6,20 , indicating that multiple gene copies are not a strict requirement for post-meiotic gene expression from the sex chromosomes. Thus, specific enhancers and transcription factors may have evolved to overcome the transcriptional repression associated with the rest of the sex chromosomes. Two potential transcription factors that facilitate post-meiotic sex-linked gene expression are Heat Shock Transcription Factor 1 (HSF1), which localizes to sex chromatin 21 and HSF2, which preferentially binds chromatin of Ypalindromic genes 22 . Consistent with this, the testis is known to have specialized transcription regulation strategies in post-meiotic testicular germ cells 23 and appears to have evolved a unique mechanism for palindromic and multicopy X-and Y-linked genes.    Table 1. B) Self-symmetry triangular dot plots of the two singleton X-palindromes to be studied, carrying the 4930567H17Rik and Mageb5 gene families, respectively. Each dot plot represents the palindromic X chromosome sequence (4930567H17Rik = chrX:70385921-70553920 and Mageb5= chrX:91624421-91790420) plotted against itself with a sliding window of 100 nucleotides (step size=1 nucleotide). When the window of 100 nucleotides is identical to the sequence it is compared to, a dot is plotted.