Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

Abstract

Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

JMJD-5/KDM8 regulates H3K36me2 and is required for late steps of homologous recombination and genome integrity

“…jmjd-5 encodes a protein of 578 aa with a C-terminal JmjC domain, present in almost all known proteins with histone demethylase activity [50]. The specificity of the catalytic activity of the mammalian homologue, JMJD5/KDM8, is controversial, with several studies reporting specific H3K36me2 demethylase activity [29, 30, 34, 51], and others reporting a possible function as a protein hydroxylase [33, 36, 52]. We therefore tested if JMJD-5 is active towards H3K36me2 by measuring the level of this mark by western blot on lysates derived from adult jmjd-5(tm3735) animals.…”

Multiparameter functional diversity of human C2H2 zinc finger proteins

“…A caveat of heterologous expression is that, by definition, it represents overexpression; thus, it is possible that an expanded range of binding events may be observed, relative to physiological levels. In a previous study, however, we found that protein induction levels were comparable to endogenous protein levels (Marcon et al 2014). This system also facilitates motif derivation; both known and novel motifs, even in repetitive sequence, can be identified on the basis of consistency with the "recognition code," which predicts binding motifs using the specificity residues (Najafabadi et al 2015a).…”

“…Cell culture conditions, sample preparation, mass spectrometry, and derivation of spectral counts were as previously described (Marcon et al 2014). We obtained confidence scores for each putative PPI using SAINTexpress (Teo et al 2014).…”

OH, the Places You’ll Go! Hydroxylation, Gene Expression, and Cancer

“…A role for Jmjd5 in regulating chromosomal segregation was also proposed to involve de-repression of Jmjd5 H3K36me2 demethylation activity by RCCD1 (Marcon et al, 2014). However, structural studies indicated that the JmjC domain of Jmjd5 is unlikely to accommodate a methylated lysyl residue, consistent with its limited homology to that of the JmjC KDM family (Del Rizzo et al, 2012;Wang et al, 2013).…”