A disproportionate impact of G9a methyltransferase deficiency on the X chromosome.

G9a is a histone methyltransferase responsible for the dimethylation of histone H3 at lysine 9 (H3K9me2). G9a plays key roles in transcriptional silencing of developmentally regulated genes, but its role in X-chromosome inactivation (XCI) has been under debate. Here, we uncover a female-specific function of G9a and demonstrate that deleting G9a has a disproportionate impact on the X chromosome relative to the rest of the genome. G9a deficiency causes a failure of XCI and female-specific hypersensitivity to drug inhibition of H3K9me2. We show that G9a interacts with Tsix and Xist RNAs, and that competitive inhibition of the G9a-RNA interaction recapitulates the XCI defect. During XCI, Xist recruits G9a to silence X-linked genes on the future inactive X. In parallel on the future Xa, Tsix recruits G9a to silence Xist in cis Thus, RNA tethers G9a for allele-specific targeting of the H3K9me2 modification and the G9a-RNA interaction is essential for XCI.

Jpx RNA activates Xist by evicting CTCF.

In mammals, dosage compensation between XX and XY individuals occurs through X chromosome inactivation (XCI). The noncoding Xist RNA is expressed and initiates XCI only when more than one X chromosome is present. Current models invoke a dependency on the X-to-autosome ratio (X:A), but molecular factors remain poorly defined. Here, we demonstrate that molecular titration between an X-encoded RNA and an autosomally encoded protein dictates Xist induction. In pre-XCI cells, CTCF protein represses Xist transcription. At the onset of XCI, Jpx RNA is upregulated, binds CTCF, and extricates CTCF from one Xist allele. We demonstrate that CTCF is an RNA-binding protein and is titrated away from the Xist promoter by Jpx RNA. Thus, Jpx activates Xist by evicting CTCF. The functional antagonism via molecular titration reveals a role for long noncoding RNA in epigenetic regulation.

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

CTCF is a master regulator that plays important roles in genome architecture and gene expression. How CTCF is recruited in a locus-specific manner is not fully understood. Evidence from epigenetic processes, such as X chromosome inactivation (XCI), indicates that CTCF associates functionally with RNA. Using genome-wide approaches to investigate the relationship between its RNA interactome and epigenomic landscape, here we report that CTCF binds thousands of transcripts in mouse embryonic stem cells, many in close proximity to CTCF's genomic binding sites. CTCF is a specific and high-affinity RNA-binding protein (Kd < 1 nM). During XCI, CTCF differentially binds the active and inactive X chromosomes and interacts directly with Tsix, Xite, and Xist RNAs. Tsix and Xite RNAs target CTCF to the X inactivation center, thereby inducing homologous X chromosome pairing. Our work elucidates one mechanism by which CTCF is recruited in a locus-specific manner and implicates CTCF-RNA interactions in long-range chromosomal interactions.

Decapping enzyme 1A breaks X-chromosome symmetry by controlling Tsix elongation and RNA turnover.

How allelic asymmetry is generated remains a major unsolved problem in epigenetics. Here we model the problem using X-chromosome inactivation by developing "BioRBP", an enzymatic RNA-proteomic method that enables probing of low-abundance interactions and an allelic RNA-depletion and -tagging system. We identify messenger RNA-decapping enzyme 1A (DCP1A) as a key regulator of Tsix, a noncoding RNA implicated in allelic choice through X-chromosome pairing. DCP1A controls Tsix half-life and transcription elongation. Depleting DCP1A causes accumulation of X-X pairs and perturbs the transition to monoallelic Tsix expression required for Xist upregulation. While ablating DCP1A causes hyperpairing, forcing Tsix degradation resolves pairing and enables Xist upregulation. We link pairing to allelic partitioning of CCCTC-binding factor (CTCF) and show that tethering DCP1A to one Tsix allele is sufficient to drive monoallelic Xist expression. Thus, DCP1A flips a bistable switch for the mutually exclusive determination of active and inactive Xs.

Exploration of CTCF post-translation modifications uncovers Serine-224 phosphorylation by PLK1 at pericentric regions during the G2/M transition.

The zinc finger CCCTC-binding protein (CTCF) carries out many functions in the cell. Although previous studies sought to explain CTCF multivalency based on sequence composition of binding sites, few examined how CTCF post-translational modification (PTM) could contribute to function. Here, we performed CTCF mass spectrometry, identified a novel phosphorylation site at Serine 224 (Ser224-P), and demonstrate that phosphorylation is carried out by Polo-like kinase 1 (PLK1). CTCF Ser224-P is chromatin-associated, mapping to at least a subset of known CTCF sites. CTCF Ser224-P accumulates during the G2/M transition of the cell cycle and is enriched at pericentric regions. The phospho-obviation mutant, S224A, appeared normal. However, the phospho-mimic mutant, S224E, is detrimental to mouse embryonic stem cell colonies. While ploidy and chromatin architecture appear unaffected, S224E mutants differentially express hundreds of genes, including p53 and p21. We have thus identified a new CTCF PTM and provided evidence of biological function.

Modulation of histone deposition by the karyopherin kap114.

The nuclear import of histones is a prerequisite for the downstream deposition of histones to form chromatin. However, the coordinate regulation of these processes remains poorly understood. Here we demonstrate that Kap114p, the primary karyopherin/importin responsible for the nuclear import of histones H2A and H2B, modulates the deposition of histones H2A and H2B by the histone chaperone Nap1p. We show that a complex comprising Kap114p, histones H2A and H2B, and Nap1p is present in the nucleus and that the presence of this complex is specifically promoted by Nap1p. This places Kap114p in a position to modulate Nap1p function, and we demonstrate by the use of two different assay systems that Kap114p inhibits Nap1p-mediated chromatin assembly. The inhibition of H2A and H2B deposition by Kap114p results in the concomitant inhibition of RCC1 loading onto chromatin. Biochemical evidence suggests that the mechanism by which Kap114p modulates histone deposition primarily involves direct histone binding, while the interaction between Kap114p and Nap1p plays a secondary role. Furthermore, we found that the inhibition of histone deposition by Kap114p is partially reversed by RanGTP. Our results indicate a novel mechanism by which cells can regulate histone deposition and establish a coordinate link between histone nuclear import and chromatin assembly.

Genetic Intersection of Tsix and Hedgehog Signaling during the Initiation of X-Chromosome Inactivation.

X-chromosome inactivation (XCI) silences one X chromosome in the female mammal and is essential to peri-implantation development. XCI is thought to be cell autonomous, with all factors required being produced within each cell. Nevertheless, external cues may exist. Here, we search for such developmental signals by combining bioinformatic, biochemical, and genetic approaches. Using ex vivo and in vivo models, we identify the Hedgehog (HH) paracrine system as a candidate signaling cascade. HH signaling keeps XCI in check in pluripotent cells and is transduced by GLI transcription factors to binding sites in Tsix, the antisense repressor of XCI. GLI potentiates Tsix expression and impedes XCI. In vivo, mutating Indian Hedgehog results in a sex ratio bias against females, and the female lethality is rescued by a second-site mutation in Tsix. These data demonstrate a genetic and functional intersection between HH and XCI and support a role for intercellular signaling during XCI.

Histone chaperones Nap1 and Vps75 regulate histone acetylation during transcription elongation.

Histone chaperones function in chromatin assembly and disassembly, suggesting they have important regulatory roles in transcription elongation. The Saccharomyces cerevisiae proteins Nap1 and Vps75 are structurally related, evolutionarily conserved histone chaperones. We showed that Nap1 genetically interacts with several transcription elongation factors and that both Nap1 and Vps75 interact with the RNA polymerase II kinase, CTK1. Loss of NAP1 or VPS75 suppressed cryptic transcription within the open reading frame (ORF) observed when strains are deleted for the kinase CTK1. Loss of the histone acetyltransferase Rtt109 also suppressed ctk1-dependent cryptic transcription. Vps75 regulates Rtt109 function, suggesting that they function together in this process. Histone H3 K9 was found to be the important lysine that is acetylated by Rtt109 during ctk1-dependent cryptic transcription. We showed that both Vps75 and Nap1 regulate the relative level of H3 K9 acetylation in the STE11 ORF. This supports a model in which Nap1, like Vps75, directly regulates Rtt109 activity or regulates the assembly of acetylated chromatin. Although Nap1 and Vps75 share many similarities, due to their distinct interactions with SET2, Nap1 and Vps75 may also play separate roles during transcription elongation. This work sheds further light on the importance of histone chaperones as general regulators of transcription elongation.

A boundary element between Tsix and Xist binds the chromatin insulator Ctcf and contributes to initiation of X-chromosome inactivation.

In mammals, X-chromosome inactivation (XCI) equalizes X-linked gene expression between XY males and XX females and is controlled by a specialized region known as the X-inactivation center (Xic). The Xic harbors two chromatin interaction domains, one centered around the noncoding Xist gene and the other around the antisense Tsix counterpart. Previous work demonstrated the existence of a chromatin transitional zone between the two domains. Here, we investigate the region and discover a conserved element, RS14, that presents a strong binding site for Ctcf protein. RS14 possesses an insulatory function suggestive of a boundary element and is crucial for cell differentiation and growth. Knocking out RS14 results in compromised Xist induction and aberrant XCI in female cells. These data demonstrate that a junction element between Tsix and Xist contributes to the initiation of XCI.

Nap1 links transcription elongation, chromatin assembly, and messenger RNP complex biogenesis.

Chromatin remodeling is central to the regulation of transcription elongation. We demonstrate that the conserved Saccharomyces cerevisiae histone chaperone Nap1 associates with chromatin. We show that Nap1 regulates transcription of PHO5, and the increase in transcript level and the higher phosphatase activity plateau observed for Deltanap1 cells suggest that the net function of Nap1 is to facilitate nucleosome reassembly during transcription elongation. To further our understanding of histone chaperones in transcription elongation, we identified factors that regulate the function of Nap1 in this process. One factor investigated is an essential mRNA export and TREX complex component, Yra1. Nap1 interacts directly with Yra1 and genetically with other TREX complex components and the mRNA export factor Mex67. Additionally, we show that the recruitment of Nap1 to the coding region of actively transcribed genes is Yra1 dependent and that its recruitment to promoters is TREX complex independent. These observations suggest that Nap1 functions provide a new connection between transcription elongation, chromatin assembly, and messenger RNP complex biogenesis.