Suppression of cryptic intragenic transcripts is required for embryonic stem cell self-renewal

Abstract

EMBO J 30 8, 1473–1484 (2011); published online March 29 2011

Recent discoveries of histone demethylases have shown that the dynamic regulation of histone methylation is important in differentiation and development. A paper in this issue of The EMBO Journal demonstrates that an H3K4me3 demethylase, KDM5B, is required for the regulation of self-renewal and pluripotency of embryonic stem (ES) cells by removing intragenic H3K4me3 and repressing cryptic transcription.

The pluripotency and self-renewal ability of ES cells has attracted great interest in uncovering the underlying mechanisms. Three transcription factors, Oct4, Sox2 and Nanog, are core regulators involved in pluripotency and self-renewal of ES cells (Boyer et al, 2005). It has been shown that the regulation of downstream targets by Oct4, Sox2 and Nanog is dependent on the epigenetic state of chromatin.

In general, methylation on histone H3K4 and H3K36 is associated with active transcription, whereas methylation on H3K9 and H3K27 is linked to gene silencing (Li et al, 2007). Gene expression is tightly regulated by enzymes mediating histone modifications. Several histone demethylases (KDMs) have been shown to be involved in cellular pluripotency. Previous studies show that KDM3A and KDM4C positively regulate self-renewal genes through demethylating repressive H3K9me2/me3 marks at their promoters (Loh et al, 2007). In this issue of The EMBO Journal, Xie et al (2011) demonstrate a novel role of KDM5B as a transcriptional activator of self-renewal-associated genes.

Through genome-wide ChIP-seq analysis, Xie et al (2011) found that KDM5B is a direct target of Nanog. Knockdown of KDM5B leads to cell differentiation and reduced ES cell proliferation. In contrast to a previous study in which KDM5B was shown to function as a repressor of genes controlling cell differentiation (Dey et al, 2008), here KDM5B functions as an activator of genes associated with self-renewal. By ChIP-seq analysis, KDM5B was found to associate with intragenic regions of genes that are positively regulated by KDM5B. Further analysis revealed that the distribution of KDM5B occupancy is highly correlated to the distribution of H3K36me3, a mark associated with actively transcribed genes, and that the demethylase is recruited to H3K36me3 though MRG15. Interestingly, the fly homologue of KDM5B, Lid, has also been found to interact with MRG15 (Lee et al, 2009). This leads to the proposal of an interesting model, in which the H3K4 demethylase crosstalks with transcription elongation-associated H3K36me3 through an Rpd3S-like histone deacetylase complex (Figure 1A). Moreover, a recent report showed that MRG15 also recruits the splicing factor, PTB, to the alternatively spliced exons marked with H3K36me3 (Luco et al, 2010), suggesting that KDM5B might also be involved in splicing by regulating intragenic H3K4me3.

Figure 1.

Model of KDM5B function in repressing cryptic transcription. (A) Intragenic histone H3K4me3 deposited along with elongating Pol II is removed by KDM5B, creating a promoter-restricted H3K4me3 enrichment. (B) In the absence of KDM5B, intragenic H3K4me3 brings transcription initiation machinery to the coding region, resulting in cryptic transcription initiating within the body of the gene.

In S. cerevisiae, H3K36me3 recruits the Rpd3S histone deacetylase complex to actively transcribed genes, thus creating a hypoacetylated chromatin environment to prevent cryptic transcription (Carrozza et al, 2005). However, the regulation of cryptic transcription in mammalian cells remains unclear. Here Xie et al (2011) found that KDM5B functions in removing H3K4me3 deposited by the elongating Pol II-associated H3K4me3 methyltransferase, MLL. KDM5B knockdown results in increased Pol II recruitment to intragenic H3K4me3 peaks, which indicates potential initiation sites of cryptic transcription (Figure 1B). In agreement with this, the depletion of KDM5B led to an increase of cryptic transcripts, whereas the level of full-length transcription is decreased. This suggests that KDM5B functions in promoting productive transcription elongation, which sustains the expression of self-renewal genes in ES cells. It is interesting that KDM5A, another member of KDM5 family, is also an MRG15-associated protein and downregulates intragenic H3K4me3 in HeLa cells (Hayakawa et al, 2007). However, in mouse ES cells, KDM5A was found to coordinate with PRC2 complex at the promoter to repress the expression of developmental genes (Pasini et al, 2008). Thus, multiple mechanisms might contribute to the specificity of the H3K4me3 demethylase recruitment to the intragenic region. The involvement of different KDMs in regulation of gene expression shows a highly specific regulatory circuit to ensure the pluripotency of ES cells.

It was known that H3K4me3 is enriched at 5′ of the gene and marks active promoters (Li et al, 2007). However, the MLL complex, which mediates H3K4 methylation, was found to interact with the elongating Pol II (Krogan et al, 2003). Here, it is proposed that KDM5B erases the H3K4me3 marks along the coding region, establishing the 5′-enriched H3K4me3 gradient. The cryptic transcription observed upon knockdown of KDM5B implicates a novel pathway, in that the initiation of cryptic transcription is a consequence of intragenic H3K4me3 deposition, which leads to the recruitment of the transcription initiation machinery to the cryptic promoter.