Packing our genes: to preserve and use

We animals – and that includes us humans – want to keep our genes, our DNA, well protected from environmental damage.  A fundamental way to do that is to wrap our 46 strings of DNA in any new cell – in total ~1 meter in length – around many small protein balls named histone octamers that consist of 8 small, basic core histones (two each of H2A, H2B, H3 and H4).  Thus many-thousand fold compacted, we can package all our genes safely in nuclei which are less than 5 μm in diameter.

However, just packaging our DNA into these nuclear particles – nucleosomes – and creating the mass of chromatin in our nuclei, prevents its use in two of the most fundamental cell processes required: DNA replication and gene transcription.

Cells must duplicate all their DNA molecules in order to be able to divide and create two daughter with a full complement of genes, a whole genome.  During this process of DNA replication, the histone octamers must be displaced, at least temporarily, at the DNA fork where DNA strands are duplicated by DNA polymerases.  Then the newly synthesized DNA must be repackaged with histones to compact and preserve our genes.  This requires the production of new histone proteins in amounts at least equal to the amounts already present.  The coordinated action of new DNA synthesis by DNA polymerases and the repackaging of the doubled amount of DNA defines the S or Synthesis phase of the cell cycle.  Animal cells can only divide the two complete daughter sets of chromosomes equally to daughter cells at mitosis and cell division if DNA duplication and repackaging was complete.  Animal cells have a dedicated gene – in multiple copies – for the replication-coupled (RC) form of histone H3: they are produced during S phase to package newly replicated DNA into nucleosomes and chromatin.

In order to make histone H3 protein, the histone H3 genes must be transcribed into RNA and translated into the H3 protein with 135 amino acids.  However, the transcription enzymes, RNA polymerases, must displace the packaging histones from the gene coding sequences and this results, at least transiently, in some loss of histone octamers because not all can be kept nearby to repackage the DNA.  Thus, animal cells need a constant supply of histones, including histone H3, because it is needed to repackage genes at all times that they are transcribed, which means 'all the time' and also in cells that no longer divide, our so-called differentiated cells.  Animals – formally all metazoans – contain a continuously expressed histone H3 gene (in multiple copies) for this specific purpose.  This replication-independent (RI) H3 histone is also known as a replacement histone because, over time and in non-dividing cells, it replaces the RC H3 form.  We know this because RI H3 protein differs in 4 of its 135 amino acids from the RC form and this allows us to separate and quantitate both forms.

The animal RI H3 form, required for proper expression of all our genes, is absolutely conserved.  When the distinct RC and RI H3 genes arose through a gene duplication event in the ancestral species of all metazoa – some 400 million years ago – the RI H3 form then created has remained unchanged and is now found in all animals, from corals to man.  The functional specialization into a replication-coupled and a transcription-linked H3 variant form has likely contributed a lot to the complexity that could develop in multicellular animals where dividing cells must coexist with differentiated ones that no longer proliferate.

A similar H3 gene duplication and functional specialization also arose in the plant kingdom, some 1 billion years ago and completely independently from the similar event in the evolution of animals.  The gene and protein differences between the replication-coupled and the transcription-linked H3 variants in plants were again just 4 amino acids, overlapping with most of the 4 differences seen in animals.  It was only after these two distinct forms of histone H3 were formed that multicellularity arose in which proliferating and differentiating cells together create the complex green organisms we call plants (Waterborg 2011b).

In those fungi that form multicellular organisms built from hyphae, often named mushrooms (formally known as basidiomycetes), but not in simpler fungi like yeast that we use to make bread and beer (formally known as ascomycetes), genome sequencing had detected by 2006 the coexistence of two distinct H3 genes in several species that differed in the same amino acids as the RC-RI pairs of H3 genes in animals and in plants.

Based on this little hint, we started in 2007 the work that led to the most recent publication.  Why did we do that?  First of all, our study of H3 gene evolution could show that these two distinct variants arose through gene duplication, independently from the animal or plant events, some 300+ million years ago (Waterborg 2011a).  So, would they behave the same as in animals and in plants?

Second: in animal and plant genomes – where cells contain at least 2 complete genomes in G1 phase and 4 in G2 phase after DNA replication – large numbers of identical gene copies of both RC and RI H3 genes exist.  If you want to study how important each variant form is, you want to make mutants and, if possible, delete each H3 variant gene to see whether cells can live with just the RC or just the RI gene – just like most of the unicellular simple eukaryotic cells can live with a single H3 form.

Third: for this study we selected Ustilago maydis, a pathogenic fungus that infects corn.  We chose it because its genome had just been sequenced and the genome contained only a single RC-like H3 gene and a single RI-like gene.  Also, we could grow it in the laboratory in haploid form, i.e. with a single genome per cell, just like yeast.  Simple.  Other researchers had developed the methods to interrupt specific Ustilago genes, or to replace sequences through homologous recombination.  This method was not as simple as in yeast but the persistent work of Anju Verma, Ph.D., yielded success.  In combination with histone H3 protein biochemistry by Jakob Waterborg, Ph.D., in cell cycle-synchronized cultures and using radioactive tracers, we got results.  (1) The predicted S-phase specific (RC) vs constitutive (RI) expression pattern of the two H3 forms was confirmed.  (2) The RI H3 form was located across transcribed genes, indicated by its post-translational acetylation state.  (3) It was subject to replacement turnover, the characteristic functional feature of all RI H3 variants in animals and in plants.

Having established the universal functional characteristics of the 2 histone H3 variants for Ustilago, which had arisen independently from similar evolutionary choices in the animal or the plant kingdoms, we found to our amazement that we could delete either H3 form and still retain viable cells! (Verma et al. 2011)

Now our continued research in Ustilago must show us how this is possible in view of the established patterns of H3 gene expression and the demonstrated functional differences between the H3 variant proteins.  If we can do this, we'll have to go on to animals and to plants and investigate what that will tell us about the apparently incompletely understood functions of histone H3 variants in building chromatin on newly replicated DNA or across recently transcribed genes.

Stay in contact!  This interesting ride is far from over!

Verma, A., Kapros, T., and Waterborg, J.H. 2011. Identification of a replication-independent replacement histone H3 in the basidiomycete Ustilago maydis. J. Biol. Chem. 286(29): 25790-25800. doi:10.1074/jbc.M111.254383.

Waterborg, J.H. 2011a. Evolution of Histone H3: Emergence of Variants and Conservation of Post-Translational Modification Sites. Biochem. Cell Biol. In Press.

Waterborg, J.H. 2011b. Plant histone acetylation: In the beginning... . Biochim. Biophys. Acta In Press. doi: 10.1016/j.bbagrm.2011.02.005.