Histone Mutation effects of Gene Expression in Lung Cells

       Histone proteins help to provide structural support for a chromosome. Each chromosome contains a long molecule of DNA and in order to fit into the cell nucleus, DNA wraps around complexes of histone proteins. Histones are known to play a role in gene regulation and access to DNA during replication and transcription. There are four histones (H2A, H2B, H3, and H4) which consist of around 102-135 amino acids. H2A commonly forms a dimer with H2B and together with H3 and H4 an octamer complex is formed. This octamer complex interacts with the negatively charged DNA to form a nucleosome. Formation of a nucleosome, chromatin remodeling, and histone-DNA interactions all can play a role in gene expression mechanisms. 

       In total, there are around 118 histone genes. Many of these genes differ by a few amino acids which is significant when studying nucleosome stability, chromatin accessibility, and gene expression. In addition, histones can be post-translationally modified. This can further effect histone stability, its interactions with DNA, and other nucleosome binding properties. Post-translational modifications are also known to be heavily related to gene expression or repression. Recent studies have shown that histone mutations could be a new class of cancer development. My research focuses on specific histone mutations and how they could contribute to tumorigenesis. 

       To start understanding the common histone mutations in cancer, studies from cBioPortal were analyzed to search all histone genes. After proper statistical methods, there were genomic alterations found in the H2A genes of 11% of cancer patients. These alterations were found in increased number of patients with many types of cancer including non-melanoma skin cancer, bladder urothelial carcinoma, and non-small cell lung cancer. These patients with genomic modifications were found to have lower survival rates compared with patients without a histone mutation. 

       Most the mutations found could be classified as missense mutations. To identify which were most common, the data from cBioPortal was compared with amino acid sequences from multiple genes that aligned. A Z-score was then calculated at each amino acid residue with any score greater than 2 recognized as a significant flashpoint of recurrent mutation. Missense mutations recognized caused a change in the charge of the amino acid or removed a positively charged amino acid. These mutational spots strongly suggest a change of function that could promote tumorigenesis. Other histone residues were found to be rarely mutated which can be attributed to specificity of DNA mutations or mutations that have a deleterious effect on cell growth, the opposite of cancer progression. 

       Focusing on the mutations found in histone H2A specifically, these made up about 20% of the histone missense mutations identified in cancer. The most common mutated H2A residue was E121. This showed decreased nucleosome sliding through ATP-utilizing chromatin remodeling and impaired yeast growth. Another common mutated H2A residue was R29, which had similar effects as E121. It demonstrated decreased nucleosome thermal stability, impaired yeast growth, and increased histone dimer exchange. It was also interesting to identify how most of the missense mutations of H2A occurred in the histone-fold domain or on an acidic patch of the histone. This acidic patch consists of six H2A and 2 H2B amino acids on the surface of the nucleosome that provides a charged surface for protein binding. Mutations that were in this region generally increased histone dimer exchange (H2A/H2B), impaired yeast growth, and decreased thermal stability. Another recent study found that the E92K mutation specifically inverts the charge of the amino acid and further supports the negative effects listed previously. 

       Another major focus is on the 25% of histone missense mutations in histone H3. The first oncohistone studied concentrated on mutations in these genes. The most mutated H3 residue is K27. As it is on the N-terminal tail, this position can be post-translationally modified by acetylation, methylation, or ubiquitination. Performing experiments to study this mutation has shown increased affinity between the histone and another sister repressive complex (PRC2). There was also decreased association with a corepressor protein (CYL) which typically functioned to connect the complex with neighboring nucleosomes. 

       This is an ongoing project that I have been working on throughout the past year. My work in the lab specifically is to help engineer a histone H2A E121K missense mutation into the endogenous histone H2A of BEAS-2B human lung epithelial cell line. With the guidance of my PI and other members of the lab, I have been able to culture and transfect cells with CRISPR ribonucleoprotein that contains a guide RNA to target a specific mutant gene sequence. After this, I help to screen cells using PCR to determine which cell line clones contain the correct mutation that we are interested in. In addition, I perform western blots to detect the epitope-tagged histone H2A. Continuing in the lab, I hope to learn how the histone mutant may disrupt chromatin structure to alter gene expression and cell phenotypes. Overall, I am keen to uncover a new mechanism of oncogenesis and play a role in the identification of new targets of cancer therapy. 

       In conclusion, histone mutations are found in many different types of cancer. They effect post-translational modifications, nucleosome stability, and chromatin compaction. Mutations in histone genes can lead to a state of gene expression that increases tumorigenesis. My research focuses on studying the different facets of histone mutations in cancer in hopes of understanding how small sequence variations of unknown significance can affect histone functional diversity.