The influence of post-translational modifications on biology of the linker histone HIS-24 in Caenorhabditis elegans.

In the eukaryotic nucleus the genome is packaged into a nucleoprotein complex known as chromatin. The chromatin is composed of DNA, core histones (H2A, H2B, H3, H4), linker histones (H1) as well as non-histone proteins. Such a specific compaction of DNA is necessary for the packaging of a very large genome of eukaryotic cells into the nucleus and it plays an essential role in the maintenance of the chromosomal integrity. On the other hand, the proper chromatin organization has a crucial function in DNA repair and in regulation of gene expression. During the transcription process the chromatin structure changes from highly condensed, ordered and inaccessible to an unfolded and genetically active form. Transition between the active and repressive chromatin states can be established, among others, by post-translational modifications (PTMs) of histones. These covalent changes are found to be important for regulation of DNA accessibility for recruitment of further chromatin effector proteins. Apart from well-known modification of the H3 and H4 core histone tails, post-translational changes of the H1 linker histone are also critical for the modulation of particular chromatin states. Linker histones (H1) are highly abundant eukaryotic chromatin proteins, which associate with linker DNA fragments, completing the chromatin fibre formation. Recent studies have reported that the post-translational methylation of the mammalian histone H1 is specifically recognised by the chromodomain of heterochromatin protein 1 (HP1). However, the exact biological role of linker histone association with HP1 has not yet been elucidated. In the present study, the physiological function of post-translational modification of the C. elegans linker histone variant HIS-24 was determined. C. elegans H1 variant HIS-24 was found to be recognised by the HP1-like proteins HPL through the mono-methylated lysine 14 of the linker histone. While mammalian HP1 protein was found to specifically interact with the di-methylated form of H1, the one of two C. elegans heterochromatin protein homologues HPL-1 was identified to bind selectively the mono-methylated form of nematode histone variant HIS-24. The transcriptional profiles analysis of his-24 and hpl deficient C. elegans animals revealed that HIS-24 and HPL-2 proteins play gene-specific roles, rather than a general repressive function. Phenotypic investigation of the C. elegans HIS-24 and HPL-2 proteins functions showed that both chromatin associated factors allow normal reproduction, somatic gonad development and vulva cell fate decision. Interestingly, the study showed that HIS-24 and HPL-2 also play a role in regulation of male tail development, and his-24 and hpl-2 loss of function mutation causes posterior transformations of mating structures in C. elegans. The genetic study of his-24 and hpl-2 mutants using a GFP-tagged reporter of two Hox genes showed ectopic expression of MAB-5 and EGL-5 homeodomain transcription factors. They are expressed in the posterior region of C. elegans and are required for development of sensory rays in male. The biochemical analyses using chromatin immunoprecipitation (ChIP) approach confirmed that expression of mab-5 and egl-5 Hox genes is negatively regulated by HIS-24. Moreover, the immunoprecipitation experiments showed that mono-methylated HIS-24K14me1 specifically binds to the H3K27me3 chromatin mark, which is associated with the Hox genes silencing. Additionally, expression of wild type form of the HIS-24::GFP but not HIS-24A14K::GFP resulted in complete restoration of male ray development, suggesting that the observed changes in male tail development depend on HIS-24 methylation. These results indicate that HIS-24K14me1 and HPL-2 may serve as essential protein components in the establishment and/or maintenance of the repressive chromatin structure at the Hox genes. In addition, the investigation of the biological function of the HIS-24 and the HP1-like proteins HPL-1 and HPL-2 revealed the cooperative transcriptional regulation of immune response-related genes. By using the genome profiling and quantitative proteome analysis approaches, the present study provides first insight into the HIS-24K14me1 and HPL-1-dependant negative regulation of genes encoding antimicrobial factors. The analysis of sensitivity to infection with gram-positive bacteria of C. elegans his-24 and hpl mutant strains showed that the absence of HIS-24 histone variant results in increased sensitivity to infection. Further, investigation of C. elegans transgenic lines revealed that especially the presence of the mono-methylated form of HIS-24 is crucial for proper innate immune response process. In addition, microscopic analysis C. elegans intestinal cells revealed that bacterial infection is also associated with changes in the localisation of HIS-24 variant. In particular, post-translationally modified form of HIS-24 alters its localisation from mostly nuclear, in uninfected nematodes, to both nuclear and cytoplasmic in the intestinal cells of infected animals. The study points to a specific immune response-related role of the C. elegans linker histone HIS-24. Furthermore, they imply a relationship between the post-translational modification of HIS-24 and the protection against penetration by microorganisms in the C. elegans organism. Taken together, the study demonstrates that the H1 linker histone is not only important for chromatin structure changes, and it should not be considered as a general repressor of transcription. Obtained results show that HIS-24 can regulate expression of specific genes and its post-translationally modified form is essential for the C. elegans male tail development as well as for the immune response modulation.