ZFP541 and KCTD19 regulate chromatin organization and transcription programs for male meiotic progression

Abstract The successful progression of meiosis prophase I requires integrating information from the structural and molecular levels. In this study, we show that ZFP541 and KCTD19 work in the same genetic pathway to regulate the progression of male meiosis and thus fertility. The Zfp541 and/or Kctd19 knockout male mice show various structural and recombination defects including detached chromosome ends, aberrant localization of chromosome axis components and recombination proteins, and globally altered histone modifications. Further analyses on RNA‐seq, ChIP‐seq, and ATAC‐seq data provide molecular evidence for the above defects and reveal that ZFP541/KCTD19 activates the expression of many genes by repressing several major transcription repressors. More importantly, we reveal an unexpected role of ZFP541/KCTD19 in directly modulating chromatin organization. These results suggest that ZFP541/KCTD19 simultaneously regulates the transcription cascade and chromatin organization to ensure the coordinated progression of multiple events at chromosome structural and biochemical levels during meiosis prophase I.


G)
Co-immunoprecipitation to show the interaction between ZFP541 and KCTD19 in testis lysates.(H) Yeast two-hybrid experiments to examine the interaction between ZFP541 and KCTD19.pGBK-p53/pGAD-T is a positive control.pGBK-ZFP541 shows self-activation.KCTD19-L, the long isoform of KCTD19; KCTD19-S, the short isoform of KCTD19.Each heterozygous male mouse was bred to one heterozygous or one knockout female.Each knockout male mouse was bred to one heterozygous female.All females were plugged.(A, B) Kctd19 -/-male mice are sterile, while Kctd19 -/-female mice show normal fertility.n = 5, 8, and 6 breeding pairs from left to right, respectively.
(C, D) Zfp541 -/-male mice are sterile, while Zfp541 -/-female mice show normal fertility.n = 8, 10, and 10 breeding pairs from left to right, respectively.(E, F) Kctd19 -/-Zfp541 -/-male mice are sterile, while Kctd19 -/- Zfp541 Figure S1.ZFP541 and KCTD19 preferentially express during spermatogenesis and interacts with each other.(A, B) RT-qPCR to show the expression of Zfp541 and Kctd19 in different tissues (A) and postnatal testis (B).Note, the two bands (variants) for Kctd19.(C, D) The schematic diagram to show the gene structures of Zfp541 (C) and Kctd19 (D).The positions of primer pair P1 and P2 for the detection of Zfp541 transcripts, and P3 and P4 for the detection of Kctd19 transcripts, are indicated.Exon 6 is absent in the short transcript of Kctd19.(E) Western blot to show ZFP541 and KCTD19 proteins in postnatal testis.(F,

Figure S2 .
Figure S2.The knockout of Zfp541 and Kctd19 is confirmed by RT-PCR and subsequent sequencing.(A) The schematic diagram of the Zfp541 knockout allele.Exon 2 and Exon 3 are deleted.(B) Two short transcripts are observed in the mutant by RT-PCR using template cDNA from 8-week testes and primers P10 and P11.(C) The DNA sequences of RT-PCR products from (B) and the predicted amino acids.The two transcripts have frameshift from the 103rd and 104th amino acids, respectively.The new stop codon (TAG/TGA) is indicated by a star (*).(D) The schematic diagram of the Kctd19 knockout allele.Two fragments within exon 2 are deleted.(E) A short transcript is observed in the mutant by RT-PCR using template cDNA from 8-week testes and primers P12 and P13.(F) The DNA sequences of RT-PCR product from (E) and the predicted amino acids.The frameshift from the 10th amino acid and the new stop codon (TGA; indicated by a star *) in the mutant allele are indicated.

Figure S11 .
Figure S10.Identified TFs and their contribution to DEGs.(A) The contribution of each candidate TF to DEGs.(B) RT-qPCR to validate the altered expression of selected TFs.The mRNA was extracted from WT and Zfp541 KO pachytene spermatocytes and the expression of selected TFs was examined by RT-qPCR.Error bar, mean ± SEM (n=3).*, p<0.05; **, p<0.01; ***, p<0.001; two-tailed Student's t-test.
Figure S13.Colocalization for ZFP541, CTCF, and RAD21.(A) Genome browser snapshot to show the localization for ZFP541, CTCF, and RAD21.(B) Venn plot to show the numbers of overlapped binding peaks between ZFP541, CTCF, and RAD21.