Heterogeneous nuclear ribonucleoprotein L Is a subunit of human KMT3a/Set2 complex required for H3 Lys-36 trimethylation activity in vivo

Abstract

The presence of histone H3 lysine 36 methylation (H3K36me) correlates with actively transcribed genes. In yeast, histone H3K36me mediated by KMT3 (also known as Set2) recruits a histone deacetylase complex, Rpd3s, to ensure the fidelity of transcription initiation. We report the purification of human KMT3a (also known as HYPB or hSet2) complex and the identification of a novel, higher eukaryotic specific subunit, heterogeneous nuclear ribonucleoprotein L (HnRNP-L). Interestingly, although KMT3a has intrinsic activity in vitro, HnRNP-L is essential in vivo. Moreover, KMT3a generates mono-, di-, and trimethylated products in vitro, but RNA interference against KMT3a or HnRNP-L down-regulates exclusively the H3K36me3 mark in vivo.

FIGURE 1.

HnRNP-L is a novel subunit of the human KMT3a complex. A, domain structure of human KMT3a and yeast KMT3. B, silver staining indicates co-elution of HnRNP-L with FLAG -KMT3a-C. Fractions were from a 2-ml Smart Superose 6 gel filtration column. The input of this column is affinity-purified KMT3a from cells stably expressing FLAG-KMT3a-C. Fraction numbers are shown at the top of the panel. C, interaction between KMT3a and HnRNP-L is independent of RNase treatment. D, endogenous, full-length KMT3a co-purifies with FLAG-HnRNP-L in nuclear extracts from stable cells expressing FLAG-HnRNP-L. E, reciprocal co-immunoprecipitation experiments demonstrate interactions between endogenous KMT3a and HnRNP-L in HeLa cells.

FIGURE 2.

Interaction domain mapping. A, left, schematic representation of GST-tagged KMT3a deletion mutants used for the interaction assay. Middle, silver stain of purified recombinant GST-KMT3a deletion mutants. Right, results from the nickel agarose pulldown assay using His-HnRNP-L. B, left, schematic representation of His-tagged HnRNP-L deletion mutants. Right, results from nickel pulldown assays using deletion mutants of His-tagged HnRNP-L and of GST-KMT3a, as indicated.

FIGURE 3.

KMT3a HKMT activity. A, KMT3a complex purified from HEK293 cells stably expressing FLAG-HnRNP-L is a nucleosomal HKMT that does not use core histones as substrates. B, KMT3a complex is specific for H3K36. C, mass spectrometric analysis showing that KMT3a has mono-, di-, and trimethylation activities in vitro.

FIGURE 4.

RNAi against HnRNP-L down-regulates H3K36me3 levels. Western analysis showing specific reduction in HnRNP-L and H3K36me3 levels in HeLa cells treated with two different siRNA pairs against HnRNP-L. A pair of siRNA with scrambled sequences was used as control.

FIGURE 5.

Immunofluorescence indicates that reduction in H3K36me3 levels correlates with reduced HnRNP-L levels in HEK293 cells transiently transfected with siRNA against HnRNP-L. siRNA-treated cells were co-stained with antibody against HnRNP-L and either histone proteins or histones containing specific modifications. Cells exhibiting reduced levels of HnRNP-L are marked by arrows. Reduced HnRNP-L levels down-regulate H3K36me3, but were ineffectual with respect to H3K36me1/2, H3K4me3, H3K9me3, H3K27me3, H3K79me2, and histone H3.

FIGURE 6.

Addition of HnRNP-L does not stimulate KMT3a activity in vitro..