Protein Complex Interactor Analysis and Differential Activity of KDM3 Subfamily Members Towards H3K9 Methylation

<div><p>Histone modifications play an important role in chromatin organization and gene regulation, and their interpretation is referred to as epigenetic control. The methylation levels of several lysine residues in histone tails are tightly controlled, and JmjC domain-containing proteins are one class of broadly expressed enzymes catalyzing methyl group removal. However, several JmjC proteins remain uncharacterized, gaps persist in understanding substrate recognition, and the integration of JmjC proteins into signaling pathways is just emerging. The KDM3 subfamily is an evolutionarily conserved group of histone demethylase proteins, thought to share lysine substrate specificity. Here we use a systematic approach to compare KDM3 subfamily members. We show that full-length KDM3A and KDM3B are H3K9me1/2 histone demethylases whereas we fail to observe histone demethylase activity for JMJD1C using immunocytochemical and biochemical approaches. Structure-function analyses revealed the importance of a single amino acid in KDM3A implicated in the catalytic activity towards H3K9me1/2 that is not conserved in JMJD1C. Moreover, we use quantitative proteomic analyses to identify subsets of the interactomes of the 3 proteins. Specific interactor candidates were identified for each of the three KDM3 subfamily members. Importantly, we find that SCAI, a known transcriptional repressor, interacts specifically with KDM3B. Taken together, we identify substantial differences in the biology of KDM3 histone demethylases, namely enzymatic activity and protein-protein interactions. Such comparative approaches pave the way to a better understanding of histone demethylase specificity and protein function at a systems level and are instrumental in identifying the more subtle differences between closely related proteins.</p></div

Enzymatic activity of KDM3 subfamily members towards H3K9 methylation.

<p>Individual KDM3 subfamily members were transiently overexpressed in U-2 OS cells. (A-M) DAPI staining indicating cell nuclei. (A'-M') Cellular expression of Avi-tagged KDM3 subfamily members, as detected by streptavidin-AlexaFluor-488 recognizing the biotinylated Avi-tag. (A’’-M’’) H3K9me1, -me2 or -me3 groups, respectively, as detected by antibody staining. White circles outline the transfected cells in the last panel of each series. Note that cells transfected with KDM3A and KDM3B (A, D, G and B, E, H) abolish H3K9me1 (A’’ and B’’) and -me2 (D’’ and E’’) but not -me3 (G’’ and H’’) staining. On the other hand, JMJD1C transfection (C, F, I) does not decrease H3K9me1 (C’’), -me2 (F’’) or -me3 (I’’) levels. The catalytic mutant versions of KDM3A(H1120A) (J, L) and KDM3B(H1560A) (K, M) neither reduce H3Kme1 (J’’, K’’) nor H3K9me2 (L’’, M’’) levels. N shows the summary of the enzymatic activity described above.</p

MS analysis of KDM3 subfamily members.

<p>(A) Phosphorylated peptides and residues identified in overexpressed KDM3A, KDM3B and JMJD1C using MS. Amino acid positions of the phosphorylated sites are indicated in the respective protein. Underlined phosphorylated sites are conserved. Potential phospho-sites within identified phospho-peptides are indicated in italics and brackets. (B) MS/MS spectra of the KDM3B peptide containing phosphorylated Y1541 (underlined). (C) Coomassie-stained gels showing affinity purifications of Avi-tagged, overexpressed KDM3 subfamily members from lysates of transfected HEK293T cells. The different lanes show individual purifications of KDM3A, KDM3B, JMJD1C C-term and JMJD1C as well as a control purification from mock-transfected HEK293T cells. The positions of the individually overexpressed proteins are indicated by orange squares, the position of the KDM3B interactor SCAI is indicated by a blue square. These samples were subjected to quantitative MS analysis. (D) Relative enrichment of KDM3B interactor candidates in relation to the mock control. The 406 proteins identified with at least 4 peptides were binned into 45 columns; stippled lines indicate 2 standard deviations from the mean. Proteins that centered around 0 were not enriched, whereas proteins retrieved on KDM3B that were enriched with ≥2 standard deviations (right stippled line) were considered KDM3B candidate interactors. KDM3B and its interactor candidate SCAI are indicated by arrows and boxed in the same color as in C.</p

SCAI is a specific interactor candidate of KDM3B.

<p>(A) SCAI protein sequence with the peptides identified by MS highlighted in red. The amino acids marked in green indicate trypsin cleavage sites. SCAI sequence coverage by MS was 51%. (B) Reciprocal co-immunoprecipitation of SCAI and KDM3B. V5-SCAI was either co-expressed with Avi-KDM3A or Avi-KDM3B. Reciprocal co-immunoprecipitations using V5- antibodies or streptavidin-coated beads were performed and the immunoprecipitated proteins from each immunoprecipitation were separated on SDS gels. A V5-antibody and streptavidin-HRP were used to detect SCAI and KDM3A or KDM3B, respectively. Only KDM3B but not KDM3A co-precipitated with and was able to precipitate V5-SCAI, respectively. (C) Sub-cellular co-localization of KDM3B and SCAI in HEK293T cells. Avi-KDM3B and V5-SCAI were co-expressed in HEK293T cells and detected by immunoreagents against their respective tags (b and c). The two proteins were found to co-localize in the nucleus (d).</p

Domain mapping of KDM3 subfamily members identifies regions important for demethylase activity towards methylated H3K9.

<p>(A) Overview of constructs used in this study (left) and summary of results obtained for each construct with regard to demethylase activity towards H3K9 and subcellular localization (right). Full-length and truncated KDM3A (a and d, respectively) and full-length KDM3B (e) show activity towards H3K9me1 and -me2. Full-length and truncated versions of JMJD1C (g and i-o, respectively) do not show any enzymatic activity against either H3K9me1 or -me2. Construct i corresponds to the alternative splice isoform 2 of JMJD1C. Note that constructs d and m as well as e and j are similar in size, respectively. The star denotes the Y to F mutation in KDM3B (f), the red box denotes the JmjC domain in each construct, the grey box denotes the putative Zinc finger. (B) Hybrid constructs in which the JmjC domain in KDM3A was exchanged with the one of KDM3B (Construct b) or JMJD1C (Construct c) were assayed for their ability to demethylate H3K9me2 and –me1. Whereas construct b was active against both –me2 and –me1, construct c was inactive against both methyl groups. The hybrid construct in which the JmjC domain in JMJD1C was exchanged with the one of KDM3A (Construct h) can neither remove methyl group H3K9me2 nor –me1; only the data for –me2 are shown for either construct. (C) MS-based assessment of KMD3A, KDM3B and JMJD1C catalytic activity towards H3K9me2 and –me1. H3K9me2 peptides were incubated for 2 hours with the required co-factors and either recombinant KDM3A (aa511-1321), KDM3B(aa879-1761) or JMJD1C (aa1696–2540). Along H3K9me2 substrate, H3K9me1 and H3K9me0 reaction products were quantified using MS. Reactions were performed in triplicates, and H3K9me0, –me1 and –me2 levels were measured at 7 time intervals during the 2 hour incubation period, hence the 21 peaks shown per sample. Note that in the case of KDM3A and KDM3B, H3K9me2 levels strongly and H3K9me1 levels weakly drop during the incubation period, while H3K9me0 levels steadily increase over the course of the experiment. Using JMJD1C, neither H3K9me0 nor –me1 were produced over time up to the end of the 2 hour incubation period, indicating that JMJD1C cannot demethylate H3K9me1 or –me2.</p