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, 9 (1), 57

Trithorax Dependent Changes in Chromatin Landscape at Enhancer and Promoter Regions Drive Female Puberty

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Trithorax Dependent Changes in Chromatin Landscape at Enhancer and Promoter Regions Drive Female Puberty

Carlos A Toro et al. Nat Commun.

Abstract

Polycomb group (PcG) proteins control the timing of puberty by repressing the Kiss1 gene in hypothalamic arcuate nucleus (ARC) neurons. Here we identify two members of the Trithorax group (TrxG) of modifiers, mixed-lineage leukemia 1 (MLL1), and 3 (MLL3), as central components of an activating epigenetic machinery that dynamically counteracts PcG repression. Preceding puberty, MLL1 changes the chromatin configuration at the promoters of Kiss1 and Tac3, two genes required for puberty to occur, from repressive to permissive. Concomitantly, MLL3 institutes a chromatin structure that changes the functional status of a Kiss1 enhancer from poised to active. RNAi-mediated, ARC-specific Mll1 knockdown reduced Kiss1 and Tac3 expression, whereas CRISPR-Cas9-directed epigenome silencing of the Kiss1 enhancer selectively reduced Kiss1 activity. Both interventions delay puberty and disrupt reproductive cyclicity. Our results demonstrate that an epigenetic switch from transcriptional repression to activation is crucial to the regulatory mechanism controlling the timing of mammalian puberty.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
TrxG gene expression in the prepubertal female rat hypothalamus. a The COMPASS families (for details see Introduction). b Expression of COMPASS genes in the MBH of prepubertal female rats assessed by RNA-seq. Data are represented as fold change between the early juvenile and the late juvenile groups (EJ/LJ). Dotted line depicts the EJ/LJ ratio of 1. Bars represent mean ±s.e.m. (n = 4). **P < 0.01 vs. EJ group; Student’s t-test. c Expression of COMPASS genes in the MBH of peripubertal female rats as determined by qPCR. RNA expression data were normalized using peptidylprolyl isomerase A as the housekeeping gene and by dividing each individual value by the average of the INF group. Bars represent mean ±s.e.m. INF = infantile (n = 7–8); EJ = early juvenile (n = 5–7); LJ = late juvenile (n = 6–8); PUB = pubertal (n = 6–9), day of the first preovulatory surge of gonadotropins. *P < 0.05; and **P < 0.01 vs. INF; one-way ANOVA followed by Student–Newman–Keuls (SNK) test
Fig. 2
Fig. 2
Mll1 and Mll3 mRNA transcripts in KNDy neurons of late juvenile female rats. a, b Abundance of Mll1 mRNA transcripts (green color) in the arcuate nucleus (ARC, dotted line) region of the MBH, as determined by FISH. 3V, third ventricle; ME, median eminence. Bars, 500 µm. ce KNDy neurons of the ARC contain Mll1 mRNA as determined by double FISH (Kiss1 mRNA, red; Mll1 mRNA, green). hj KNDy neurons also contain Mll3 mRNA. Bars, 100 µm. f, g Lack of hybridization in sections incubated with a sense  Mll1  RNA probe. k, l Lack of hybridization in sections incubated with a sense Mll3 RNA probe. Bars, 200 µm. s = sense; as = antisense
Fig. 3
Fig. 3
Kiss1, Tac3, and Pdyn mRNA expression, and promoter chromatin state in the prepubertal female rat hypothalamus. a Expression of Kiss1, Tac3, and Pdyn mRNA in the MBH of prepubertal female rats as determined by qPCR. Bars represent mean ±s.e.m. (INF, EJ, and LJ; n = 8); AU = arbitrary units. RNA expression data were normalized by dividing each individual value by the average of the INF group. b Changes in MLL1 recruitment to the Kiss1, Tac3, and Pdyn promoters before puberty. c Changes in MLL3 recruitment to the Kiss1, Tac3, and Pdyn promoters before puberty. d, e Abundance of the TrxG-dependent activating marks H3K4me2 (d) and H3K4me3 (e) during prepubertal development. Insets in e depict H3K4me3 content at the Kiss1 and Tac3 promoters in the CTX during prepubertal development. f EED recruitment to the Kiss1, Tac3, and Pdyn promoters before puberty. g Abundance of the PcG-dependent repressive mark H3K27me3 at the Kiss1, Tac3, and Pdyn before puberty or at the time of the first preovulatory surge of gonadotropins (late proestrus, LP) at the Kiss1 and Tac3 promoters (insets in g). h Abundance of the PcG-independent repressive mark H3K9me3 at the Kiss1, Tac3, and Pdyn promoters before puberty. Bars represent mean ±s.e.m. (INF, EJ, and LJ; n = 5–8; insets in e; n = 4; insets in g; n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. INF; one-way ANOVA followed by SNK test
Fig. 4
Fig. 4
Effect of MLL1 and EED on KISS1, TAC3, and PDYN promoter (p) activity. Bars represent mean ± s.e.m. (n = 6). ***P < 0.001 vs. all other groups; **P < 0.01 vs. Kiss1 promoter alone; *P < 0.05 vs. Tac3 promoter alone; one-way ANOVA followed by SNK test
Fig. 5
Fig. 5
Silencing the Mll1 gene in the ARC of immature female rats. a Lentiviral construct (LV-shMll1-GFP) encoding sh2612 that most effectively silences Mll1. b Transduction of ARC cells with sh2612 delivered to the ARC of EJ female rats. Green: sh2612 infected cells; blue: Hoechst-stained cell nuclei in the MBH; dotted line: ARC. Bars, 200 µm. c Cumulative percent day at vaginal opening (VO), and d cumulative percent day at first estrus (FE) in control (C; n = 9) and sh2612 (n = 7)-injected rats. Blue shade depicts the time when all C animals show VO or FE. e Percent of time spent in different stages of the estrous cycle by rats injected with C (n = 9) or sh2612 (n = 7). f Examples of estrous cycle patterns in rats injected with C or sh2612. g Mll1, Kiss1, Tac3, and Pdyn mRNA levels detected by qPCR in the ARC of LJ female rats receiving C (n = 8) or sh2612 (n = 8). h Expression of puberty-inhibitory or i puberty-activating genes in the ARC of LJ female rats receiving C (n = 8) or sh2612 (n = 8). RNA expression data were normalized by using peptidylprolyl isomerase A as the housekeeping gene and dividing each individual value by the average of the C group. P = proestrous; E = estrous; ED = transitional phase (estrous/diestrous); D = diestrous; AU = arbitrary units; 3V = third ventricle. Bars represent mean ±s.e.m. (**P < 0.01, ***P < 0.001 vs. C, Student’s t-test)
Fig. 6
Fig. 6
A genomic region upstream of the Kiss1 gene behaves as a puberty-activated enhancer domain. a ChIP-seq tracts of H3K4me1 and H4K27ac within 12 kb of Kiss1 5′-flanking region detected using ARC chromatin from LJ female rats. TSS = transcription start site (ENSRNOT00000077054.1). ENCODE regions of DNase I hypersensitivity and p300/CBP binding from mouse forebrain are also shown (green and gray boxes, respectively). Two genomic regions (site 1 and site 2, shaded blue) enriched for H3K4me1 and H3K27ac were identified as putative enhancers of the Kiss1 gene. b Abundance of H3K4me1 (dark green), H3K27ac (red), p300/CPB (red), MLL3 (light green), MLL4 (orange), MLL1 (yellow), EED (dark blue), and H3K27me3 (light blue) at enhancer sites 1 and 2 in the ARC throughout prepubertal development. Bars represent mean ±s.e.m. (n = 6–8 per group). *P < 0.05 and **P < 0.01 vs. INF; one-way ANOVA followed by SNK test
Fig. 7
Fig. 7
CRISPR-dSaCas9-KRAB-mediated epigenome silencing of the ARC Kiss1 enhancer. a Schematic representation of the AAV-dSaCas9-KRAB construct used for epigenome editing of the Kiss1 enhancer. The AAV plasmid backbone contains a CMV promoter driving expression of dSaCas9-KRAB, two nuclear localization signals (NLS) flanking dSaCas9, and three copies of an influenza hemagglutinin (3xHA) tag. It also contains a single-guide (sg) RNA cloning site and a U6 promoter driving sgRNA expression. sgRNAs complementary to five different sequences in site 1, the putative Kiss1 enhancer domain, were identified using an online tool (https://www.deskgen.com/) and cloned into the AAV-dSaCas9-KRAB vector as recommended. b H3K9me3 content at the Kiss1 enhancer (site 1) or c Kiss1 promoter as assessed by ChIP-qPCR 2 days after transfecting Rat1 cells with AAV-dSaCas9-KRAB-sgRNAs 1–5 (sg1–5) or either of two control constructs, AAV-dSaCas9-KRAB (CK) or AAV-dSaCas9 (ΔK) (n = 3, experiment run in duplicate). d MLL3 and EED occupancy of the Kiss1 enhancer site 1 in cells transfected with CK or dSaCas9-KRAB-sgRNA4 (sg4) (n = 3, experiment run in duplicate). e, f H3K27ac content at the Kiss1 enhancer site 1 (e) and Kiss1 promoter (f) in CK and sg4 transfected cells (n = 3, experiment run in duplicate). g Kiss1, Tac3, and Pdyn mRNA expression in Rat1 cells transfected with either CK (n = 6), ΔK (n = 4), sg1 (n = 6), or sg4 (n = 8). RNA expression data were normalized by using peptidylprolyl isomerase A as the housekeeping gene and dividing each individual value by the average of the CK group. ChIP-PCR data were also normalized by dividing each individual value by the average of the CK group. Bars represent mean ±s.e.m. (in b, c, g: ***P < 0.001 vs. control (CK) cells; one-way ANOVA followed by the Dunnett’s test; in df: **P < 0.01 vs. control (CK) cells; Student’s t-test). AU = arbitrary units
Fig. 8
Fig. 8
KRAB-sgRNA-mediated epigenome editing of the ARC Kiss1 enhancer in the ARC of immature female rats. a dSaCas9 mRNA content in the ARC as compared with the lateral hypothalamus (LH) in rats microinjected bilaterally with sg4 or CK targeting the ARC. b Cumulative percent of animals showing vaginal opening (VO), and c first estrus (FE) in control CK or sg4-injected rats. Blue shade represents the time when all C animals had VO or FE. d Percent of time that rats injected with CK or sg4 spent in different stages of the estrous cycle. e Examples of estrous cycle patterns in rats injected with CK or sg4. f Kiss1, Tac3, and Pdyn mRNA levels in the ARC of animals injected with CK or sg4 detected by qPCR. g Expression of puberty-inhibitory genes (blue bars) or puberty-activating genes (magenta bars) in the ARC of female rats receiving CK or sg4. RNA expression data were normalized by using peptidylprolyl isomerase A as the housekeeping gene and dividing each individual value by the average of the CK group. P = proestrous; E = estrous; ED = transitional phase (estrous/diestrous); D = diestrous; AU = arbitrary units (n = 7–8 per group). Bars represent mean ±s.e.m. (**P < 0.01, ***P < 0.001 vs. CK-injected controls, Student’s t-test)

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