Histone methylation and the recently identified histone demethylases play important roles in gene transcription regulation. The first identified histone demethylase LSD1/AOF2/KDM1a was originally characterized as a transcriptional repressor, functioning in part by removing active H3K4me2 marks from promoter regions. Here we describe an important function of human LSD2/AOF1/KDM1b in active gene transcription. LSD2 shares similar substrate specificity with LSD1 and demethylates mono- and di-methylated H3K4. Instead of functioning as a co-repressor, LSD2 is important for optimal gene transcription. LSD2 is unique in associating specifically with the gene bodies of actively transcribed genes, but not at promoters. A specific function of LSD2 is to maintain low levels of H3K4 methylation within elongation regions. Furthermore, LSD2 forms complexes with H3K9 and H3K36 methyltransferases. These histone modification enzymes together orchestrate appropriate histone modifications in order to maintain a repressive chromatin structure at elongation regions, which may be important for optimal transcription elongation.
During the preparation of this manuscript, the enzymatic activity and functional studies of LSD2/AOF1 were reported by others (Ciccone et al., 2009
; Karytinos et al., 2009
; Yang et al., 2010
). Work by Ciccone et al has shown that LSD2 knockout mice fail to establish a subset of maternal imprints. These results likely reflect the functional relationship between DNA methylation and H3K4 methylation, although the mechanism underlining how LSD2 directly regulates DNA methylation has yet been discovered. It is noted that this finding is confined to a particular stage of oocyte maturation and a small subset of imprinted genes, while global DNA methylation is unchanged. To explore the function of LSD2 outside of oocytes, we examined more than 90 genes in HeLa cells after LSD2 depletion (including LSD2 targets and a collection of cancer-related genes) and found no significant alterations in DNA methylation on the CpG islands of corresponding promoters (data not shown). These findings indicate multiple functions of LSD2 at different stages of development.
What is the function of LSD2 in somatic cells, given that LSD2 expression persists post-development? A study by Yang et al suggests a functional role of LSD2 in direct regulation of gene transcription, mainly towards transcriptional repression, albeit independent of its own H3K4 demethylase or HDAC activity (Yang et al., 2010
). However, it is worth noting that this study failed to demonstrate dosage-dependent effects on reporter gene activity, or identify any endogenous targets of LSD2. On the other hand, we observed a predominant down-regulation of transcription upon depletion of endogenous LSD2, consistent with a positive role for LSD2 in transcription.
The positive role of LSD2 in transcription regulation may at first seem to be at odds with its innate enzymatic activity, which removes H3K4me2/1 marks of active genes. Nonetheless, the incorporation of canonical `repressive' marks into regions of active transcription does represent a newly appreciated and intriguing development. In yeast, Eaf3 of Rdp3 complex binds to tri-methylated H3K36 and recruits HDAC activity to the loci (Carrozza et al., 2005
; Joshi and Struhl, 2005
). It is proposed that this HDAC activity counteracts the spreading of acetylation from promoters and is required to maintain repressive state of chromatin after elongating Pol II. It is tempting to speculate that an LSD2-H3K4me2 relationship within gene bodies at least in part resonates with the aforementioned role of HDAC-acetylation on active transcription. Interestingly, we did not detect any HDAC activity from the LSD2 complex; nor did we detect any changes in intragenic acetyl-H3 levels upon LSD2 depletion. It is therefore probable that LSD2 function in active gene transcription in mammals is independent of HDAC activity. Instead, it may provide a complementary epigenetic mechanism for maintaining co-transcriptional `repressive' chromatin state.
In addition to H3K4 demethylase activity, LSD2 also contributes to maintaining an optimal `repressive' environment of actively transcribed regions through its interaction with H3K9 methyltransferase G9a. We show that G9a forms stable complexes with LSD2 and regulates H3K9 methylation at LSD2 binding sites within coding regions, and is required for active gene transcription. Consistent with our findings, it has also been reported that G9a binds within actively transcribed genes and can function as a coactivator of nuclear receptors (Lee et al., 2006a
), in addition to its well known role as a repressor (Roopra et al., 2004
). It is plausible that in mammals, beside HDACs, LSD2 and G9a provide additional layers of control of repressive chromatin structure of elongating chromatin that could be important for efficient and faithful transcription.
Additional factors present in the LSD2 complex may also shed light on the mechanism by which LSD2 positively regulates transcription. We observed and validated the presence of pTEFb in purified LSD2 complex, as well as Ser-2 phosphorylated RNA polymerase. The functional link between elongating Pol II and LSD2 is further supported by our observation that LSD2 depletion impedes the full induction of immediately early responsive genes, such as EGR-1, which are known to be regulated post-initiation, at the level of elongation (Wang et al., 2005
We note that not all of the LSD2 associated genes identified by ChIP-chip were dramatically affected by LSD2 depletion, despite robust increases of H3K4me2 levels of at the LSD2 binding regions. We believe that this finding is not unexpected. In fact, inactivation of other transcriptional elongation-associated histone modifying enzymes, such as the deacetylase Rpd3 or H3K36 methyltransferase SETD2, also cause only moderate transcriptional changes in a subset of its associated genes in both yeast and C. elegans
(Edmunds et al., 2008
; Sharma et al., 2007
; Wang et al., 2002
). This could be explained in part by the understanding that transcription elongation is regulated at multiple layers, by post-translational modification of the CTD of the Pol II controlled by both positive (p-TEFb) or negative (NELF) elongation factors, and finally chromatin remodeling and modifying enzymes (Core and Lis, 2008
; Sims et al., 2004
). Each layer of regulatory factors likely differentially and perhaps cooperatively contributes to the transcriptional outcomes of specific genes, providing flexibility and fidelity to ensure proper programs of gene expression essential for cellular and biological processes.
It is of great interest to find several PWWP-domain containing proteins in association with LSD2 complex, including the H3K36 methyltransferase NSD3; MSH6, a component of DNA mismatch repair machinery; and an uncharacterized protein NPAC. The PWWP domain is a loosely conserved protein module found in eukaryotic nuclear proteins, which are often associated with chromatin (Maurer-Stroh et al., 2003
). Recently, specific recognition of histone methylation marks by PWWP domains has been reported (Vezzoli et al., 2010
; Wang et al., 2009
). NSD3 is a major component of the LSD2 complex, and demonstrates a strong biochemical intereaction ( and Figure S6B
). We detected NSD3 binding to a small subset of LSD2 targets by ChIP (Figure S6E
) and observed a moderate effect of NSD3 depletion on the expression of LSD2 target genes (Figure S6F
). It is possible that NSD3 and LSD2 form complexes in vivo
and coordinate the dynamics of H3K4 and H3K36 methylation for transcription elongation of certain genes; however further investigation is required to fully define the nature of this interaction. We are tempted to speculate that PWWP domain-containing factors in LSD2 complex may function in LSD2 targeting through binding to specific histone methylation marks. Future investigation into these possibilities is warranted to further our understanding of the role PWWP- containing proteins in the recruitment of LSD2 and their potential in regulating LSD2 functions.
Human LSD2 is a candidate cancer-related gene located at chromosome 6p22, a genomic region with high incidence of chromosomal translocations, deletions or amplifications in multiple cancer types (Heidenblad et al., 2008
; Orlic et al., 2006
). Furthermore, data from the ONCOMINE cancer database shows that human LSD2 levels are significantly lower in some leukemias, seminomas, and a few classes of ER-negative breast cancers. These correlations suggest that the biological function of LSD2 and its exact role in human cancer are likely tissue or cell type dependent. The function of LSD2 in transcription regulation may provide insight to the epigenetic mechanism involving gene regulation in tumorigenesis, therefore offering a molecular basis for future development of demethylase inhibitor-based anti-cancer drugs.