Downregulation of X-linked gene expression during C. elegans dosage compensation allows study of gene expression mechanisms that act over large chromosomal regions. Previous studies have identified a condensin-like complex and other chromatin-associated proteins required for this process, but the mechanism by which these proteins lower X-linked gene transcription is not known. Here we show that the DCC generates X-linked enrichment of the post-transcriptional histone modification H4K20me1 and that this modification is important for dosage compensation.
As we observed here for C. elegans
, H4K20me1 in other organisms is enriched on gene regions and its level is positively correlated with gene expression 
. Although H4K20me1 levels are highest on actively transcribed genes, functional experiments in vertebrates and Drosophila
point to a repressive role in gene expression. Knockdown of the H4K20me1 methyltransferase Pr-Set7 in human cells caused a two-fold upregulation of genes normally harbouring H4K20me1 
, and mutation of Pr-Set7 in Drosophila
leads to position effect variegation, a hallmark of genes required for heterochromatic gene repression 
. H4K20me1 is also associated with the inactive X chromosome during X inactivation in vertebrates 
. Therefore, the high levels of H4K20me1 we observed on the C. elegans
X chromosome are consistent with a role in dosage compensation-mediated repression of gene expression.
What is the mechanism that leads to higher H4K20me1 levels and lower H4K20me3 levels on the hermaphrodite X chromosomes relative to autosomes? Our results suggest that this is achieved at least in part through DCC inhibition of SET-4 activity on X. Lower conversion of H4K20me1 to H4K20me2/3 by SET-4 on the X chromosome would lead to relatively higher H4K20me1 levels and lower H4K20me2/3 levels there. Several observations support this model. First, dosage compensation mutants show lower overall H4K20me1 levels and higher H4K20me3 levels compared to wild type. Second, the lower overall H4K20me1 level in DCC mutants is due to inappropriate SET-4 activity, supporting the idea that active dosage compensation inhibits SET-4 activity. Third, the difference in H4K20me1 levels on the X versus the autosomes is abolished in set-4 mutants, indicating a role for SET-4 in generating the asymmetry. We propose that a component of the DCC prevents SET-4 from acting on the X chromosome, leading to maintenance of H4K20me1 on X, whereas H4K20me1 is preferentially converted to H4K20me2/3 on the autosomes. Because H4K20me1 levels are similar on X and autosomes in set-4 mutants, SET-1 might be equally active in generating H4K20me1 on all chromosomes.
Our results suggest that of the three H4K20 methylation states, H4K20me1 is the key modification for dosage compensation. Whereas loss of dosage compensation leads to lethality of XX embryos, set-4 null mutants, which have strongly reduced levels of H4K20me2 and H4K20me3, are viable, and RNAi of set-4 does not enhance lethality of DCC mutants. This suggests that these modifications are not necessary for dosage compensation. In contrast, RNAi depletion of maternal and zygotic set-1 leads to loss of H4K20me1 and embryonic lethality, set-1 genetically interacts with dosage compensation mutants, and set-1 mutants show upregulation of X-linked gene expression.
When does H4K20me1 function in dosage compensation? The DCC is recruited to the X chromosome around the 30-cell stage whereas X-chromosome enrichment of H4K20me1 occurs several hours later. This difference in timing suggests that there might be two separable aspects of dosage compensation during embryogenesis, for example initiation and maintenance. Although the DCC is recruited to the X in early embryogenesis, it is not yet known when repression of X-linked gene expression is initiated. The DCC might be active immediately after recruitment or might become active later in embryogenesis. Furthermore, although H4K20me1 becomes highly enriched on X in late embryogenesis, it is possible that a basal level on the X chromosome is functional earlier. Because the DCC component DPY-27 shows apparently normal localization to the X chromosome in the absence of SET-1 or SET-4, H4K20me1 does not appear to be a recruitment signal for the DCC. Instead, H4K20me1 may be important for the function of the DCC in downregulating gene expression. Key future questions to address are when during embryogenesis X-linked gene expression is initially downregulated, and when H4K20me1 function is necessary.
Regulation of histone modification levels also occurs during dosage compensation in other organisms. For example, in Drosophila, where gene expression on the single X chromosome in males is upregulated two-fold to match that of the two X chromosomes in females, dosage compensation acts to increase H4K16ac levels on the single male X. In addition, the inactive X chromosome of female mammals displays high levels of several histone modifications, including H4K20me1 
. H4K20me1 enrichment correlates with Xist expression, is independent of transcriptional silencing, and marks the early steps of X inactivation 
In addition to the strong enrichment of H4K20me1 on the X chromosome in C. elegans
, Liu et al. showed that several marks of gene activity, including H4K16ac, were lower on X linked genes than on autosomal genes 
. Using immunofluorescence assays on gut nuclei, a recent report by Wells et al. showed that the X/A difference in H4K16ac levels depends on dosage compensation and on sir-2.1
, a putative H4K16 deacetylase 
. It is not clear if H4K16Ac plays a role in dosage compensation as depletion of sir-2.1
did not genetically interact with a DC mutant. The enzyme that generates H4K16Ac is not yet known.
Using immunofluorescence assays, Wells et al. also observed that H4K20me1 enrichment on X is dependent on dosage compensation, and on SET-1 and SET-4 
. Our immunofluorescence results are broadly similar, and our ChIP experiments give a higher resolution view, strengthening these conclusions. In support of a role for methylation of H4K20 in dosage compensation, Wells et al. observed that simultaneous reduction of set-1
by RNAi could rescue mutant males that normally die due to active dosage compensation. However, the H4K20 methylation state was not determined after simultaneous depletion of set-1
, so the specific alteration of methylation of H4K20 that caused rescue is not known. The reported X/A differences in H4K16ac levels also depended on set-1
, suggesting that H4K16Ac might be regulated by H4K20 methylation state. Although the exact mechanisms of dosage compensation vary, studies in different organisms suggest that global regulation of H4K16ac and H4K20me1 levels might be a conserved feature of these chromosome-wide gene regulation mechanisms.
Our results indicate that H4K20me1 is important for repression of X-linked gene expression. How might H4K20me1 function in transcriptional repression? Several links in the literature suggest roles for H4K20me1 in chromatin compaction. For example, the Malignant-Brain-Tumor (MBT) domains of human L3MBTL1 compact nucleosomal arrays by recognizing mono and dimethylation of H4K20 and H1bK26 
. Furthermore, L3MBTL1 has transcription repressor activity that is enhanced by Pr-Set7, and its chromatin association depends on H4K20me1 
. It is not yet known whether C. elegans
MBT repeat proteins LIN-61 or MBTR-1 are involved in dosage compensation or bind H4K20me1.
H4K20me1 has also been shown to be important during mitosis. H4K20me1 levels are high on mitotic chromatin (
and this study), and in mammalian cells inhibition of Pr-Set7 leads to defects in cell cycle progression 
. Although the function of H420me1 in cell cycle progression is not yet understood, a key aspect of the loss of function phenotype is reduced chromosome compaction. Furthermore, a recent study demonstrated that two components of condensin II, N-CAPD3 and N-CAPG2, can directly bind H4K20me1 
. This raises the exciting possibility that condensin IDC
might function to compact chromatin through binding H4K20me1. Increased compaction of the X chromosome relative to the autosomes might reduce access by RNA polymerase, leading to lower X-linked gene expression. Consistent with this idea, DCC mutants were recently shown to have increased RNA polymerase II levels on the X 
. We propose that condensin complexes and H4K20me1 might be intimately linked in diverse chromatin-regulating events.