Evolutionary Insights into H2A.Z Function in Gene Regulation

DNA provides instructions for how cells should be built and run, yet there are two fundamental problems: First, the way one cell nucleus can fit the whole DNAs length without entanglement might seem surprising at first sight. Second, the DNAs features-or genesare complex and often have contradictory functions. To solve these problems, eukaryotic organisms, from unicellular algae to multi- tissue plants and animals, wrap their DNA into compact bundles around a group of proteins called histones, making structures called nucleosomes. The four core histone families H2A, H2B, H3, and H4 act together to regulate DNAs replication and repair during proliferation and cell division, as well as its transcription into RNA when genes are expressed. Although the structure of nucleosomes and their histones is universal, eukaryotic genomes contain dozens of copies of each core histone family, which often differ in their function. For nearly 30 years the function of these so-called variant histones has been investigated, yet to date, we lack a fundamental understanding of why there are so many of them and what the evolutionary forces that shaped their emergence were. Here, I investigate the molecular evolution of one histone variant, H2A.Z, to understand its function in regulating the genome. Genes encoding H2A.Z have been found in nearly every eukaryote, and in these cases, H2A.Z is often essential for cell survival. Further, H2A.Z has been investigated in various organisms, where it was shown to both increase and decrease the expression of genes. This duality is unusual, and despite decades of work, H2A.Zs function remains difficult to interpret. A key challenge in studying histone variants and their function has been the sheer complexity of the cellular systems surrounding nucleosomes. Instead of a single gene encoding a single function, histones are multifunctional, meaning that perturbing them has pervasive consequences throughout the cell, complicating interpretations. To bypass this limitation, I have created an experimental system based in the yeast Schizosaccharomyces pombe where H2A.Z genes from across the tree of life have been introduced, generating a series of lines that differ vastly in their gene transcription. Leveraging this variation, I will characterize what defects or altered functions in transcription exist. This will allow me to conclude (1) what features of H2A.Z are important for its function in transcription, and (2) which interactors might drive these differences. Thus, this approach moves beyond the fundamentally correlative methods that have been used to date to study histone variants. In a larger context, understanding the evolution of H2A.Z and its function will shed light on the fundamental molecular mechanisms leading to transcriptional diversity and hint at how universal biological systems like histones and nucleosomes can give rise to the stunning diversity of multicellular life.

Inside every eukaryotic cell, meters of DNA is tightly packed to fit inside the central hub of the cell, the nucleus. This packaging isn't just for storage; it acts like a sophisticated regulatory system allowing cells to respond to the needs of their environment, whether in development or in response to external stress. The DNA is wrapped around protein complexes called nucleosomes that are composes of a special class of proteins, histones. Whereas most nucleosomes are fairly uniform in composition, cells deploy special versions referred to as histone variants to specific genes with distinct functions. One of the most important of these is called H2A.Z, yet although it was first described 30 years ago, and is essential for life, its importance for the workings of the genome has remained largely unresolved. This work aimed to determine the molecular mechanisms underlying H2A.Z's importance for regulating cells, particularly linking its control of a process called gene transcription to its impact on physiology. We focused on three aspects of its function, investigating how its location in the genome changes its behavior, which parts of its structure are responsible for its unique functions, and which helper proteins in the cell recognize H2A.Z to help turn genes on or off. To study this, we used a synthetic biology approach. We took H2A.Z sequences from species across the eukaryotic tree of life, expressing them in a living host cell. This allowed us to see how different versions of this protein worked in the cell, and to identify what molecular cause for differences in how the cell read its genetic instructions and how those changes affected the cell's overall health and growth. Contrasting long-standing models for its function, we discovered that H2A.Z isn't just a passive structure for packaging DNA. Instead, it acts like a physical signal placed within genes to directly instruct the machinery responsible for reading genes, telling it how fast or slow to go. Moreover, we identified a new control region of the protein, a largely overlooked loop region that is critical for setting this function, and that evolution has used repeated to diversify H2A.Z's function. Finally, we established a programmatic molecular logic linking this loop region to its broader role in regulating the cell's physiology. This work answers a long-standing question in the field of chromatin biology about how cells regulate their genes. Further, the insights we gained here have allowed us to create a new technological platform. By understanding how to program these DNA spools, we can better engineer cells for use in medicine and biotechnology, creating more efficient ways to produce therapeutic agents or sustainable materials.