CXXC finger protein 1 restricts the Setd1A histone H3K4 methyltransferase complex to euchromatin

Abstract

CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is a component of the euchromatic Setd1A histone H3K4 methyltransferase complex, and is a critical regulator of histone methylation, cytosine methylation, cellular differentiation, and vertebrate development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1−/−) are viable but show increased levels of global histone H3K4 methylation, suggesting that Cfp1 functions to inhibit or restrict the activity of the Setd1A histone H3K4 methyltransferase complex. The studies reported here reveal that ES cells lacking Cfp1 contain decreased levels of Setd1A and show subnuclear mislocalization of both Setd1A and trimethylation of histone H3K4 with regions of heterochromatin. Remarkably, structure–function studies reveal that expression of either the N‐terminal fragment of Cfp1 (amino acids 1–367) or the C‐terminal fragment of Cfp1 (amino acids 361–656) is sufficient to restore appropriate levels of Setd1A in CXXC1−/− ES cells. Furthermore, functional analysis of various Cfp1 point mutations reveals that retention of either Cfp1 DNA‐binding activity or association with the Setd1 histone H3K4 methyltransferase complex is required to restore normal Setd1A levels. In contrast, expression of full‐length Cfp1 in CXXC1−/− ES cells is required to restrict Setd1A and histone H3K4 trimethylation to euchromatin, indicating that both Cfp1 DNA‐binding activity and interaction with the Setd1A complex are required for appropriate genomic targeting of the Setd1A complex. These studies illustrate the complexity of Cfp1 function, and identify Cfp1 as a regulator of Setd1A genomic targeting.

Efficient differentiation of murine embryonic stem cells requires the binding of CXXC finger protein 1 to DNA or methylated histone H3-Lys4

“…Similar to previous observations [25], the percent co-localization of histone H3K4me3 with DAPI-bright heterochromatin was significantly increased in CXXC1 -/-ES cells (22.1%) and CXXC1 -/-ES cells carrying the empty expression vector (21.9%) compared to that found in CXXC1 +/+ ES cells (3.5%) or CXXC1 -/-ES cells rescued with the WT Cfp1 expression vector (3.3%). Importantly, a rescue of proper restriction of histone H3K4me3 to euchromatic sub-nuclear domains was observed in CXXC1 -/-ES cells expressing the W49A form of Cfp1 (4.6%) (Fig.…”

“…Cfp1 is required to restrict the sub-nuclear localization of Setd1 and histone H3K4me3 to euchromatin domains [25]. Moreover, Cfp1 also regulates the levels of the H3K4me3 mark at many CGI-rich gene promoters, and the DNA-binding function of Cfp1 is surprisingly not necessary for this function [27].…”

“…This defect in sub-nuclear targeting of the histone H3K4me3 mark is rescued by expression of Cfp1 in CXXC1 -/-ES cells, but not by mutated forms of Cfp1 that lack either DNA-binding activity (C169A) or the ability to associate with the Set1 complex (C375A) [25].…”

“…In the absence of Cfp1, the Setd1a complex and its product, histone H3K4me3, are partially mis-localized to DAPIbright heterochromatin as observed by confocal microscopy. This suggests that Cfp1 restricts the Setd1a complex to euchromatin [25]. Furthermore, chromatin immunoprecipitation studies revealed that Cfp1 is localized at ~80% of CpG islands (CGIs), and is required for deposition of histone H3K4me3 marks at CGIs [26] [27].…”

Dynamic acetylation of all lysine-4 trimethylated histone H3 is evolutionarily conserved and mediated by p300/CBP

“…55) may primarily recruit CXXC motif-containing proteins, including the H3K4 methyltransferase MLL1 (56) and CGBP/ Cfp1, which associates with H3K4 methyltransferases (57,58). DNA binding by Cfp1 has recently been shown to restrict Setd1A and H3K4me3 to euchromatic nonmethylated CpG regions (59). Similarly, ChIP-seq analysis has shown a tight association between Cfp1 and H3K4me3 at CpG islands, and Cfp1 knockdown depletes H3K4me3 levels at nonmethylated CpGs (60).…”