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In vitro characterization of Gene Regulatory Innovations in the Human Genome


The Human Genome Project has generated 3 Giga base pairs (bp) of DNA that can be visualized and analyzed on the UCSC Genome Browser. A major challenge in the post-genomics era is to functionally annotate this map of our genome. Uniquely human features at the DNA level can be interrogated and likely hold the secrets to human evolution of specific traits as well as disease. When comparing the human genome to related primate or mammalian genomes two major classes change are: single base pair substitutions and larger structural rearrangements such as insertion of new DNA. During my thesis work I investigated in detail examples of each.

Human Accelerated Region 1 (HAR1) is defined as the region of the human genome most significantly changed since we diverged from chimpanzees as it contains 18 substitutions in a 118bp region that has been conserved in our genomes from chicken to chimpanzee at 96% identity. This small genomic region adopts a stable RNA secondary structure and is included in a long non-coding RNAs expressed in during neural development. I used molecular, genetic and biochemical methods to assign HAR1 RNAs a cellular function, and to test the hypothesis that the HAR1 containing RNAs contribute to human specific features of brain development.

Transposable elements (TEs) are an important source of gene regulatory elements and are abundant and active in our genome, but new insertions can be deleterious to the host. Like integrated viral DNA, TEs are transcriptionally silenced by the host, and must evolve to evade host transcriptional silencing mechanisms to maintain their ability to mobilize. KRAB domain Zinc Finger Proteins (KZNFs) are candidate transcription factors for recognizing viral or mobile element DNA and recruiting transcriptional repression complexes in primates. Humans have ~150 primate-specific KZNFs that could recruit KAP1-corepressor complex through their KRAB domain to target active or recently active mobile elements for transcriptional repression. We show that human KZNFs ZNF91 and ZNF93 bind and transcriptionally repress two prominent mobile element types that have recently expanded in apes: SVA and L1 mobile elements, respectively. We demonstrate that human ZNF93 recognizes L1PA4, and show that in the last common ancestor of humans and gorilla, a new family of L1, L1PA2, evaded this response by loss of the ZNF93 binding site. Ancestral reconstruction of the ZNF91 locus shows two duplication events that added 7 Zinc fingers to ZNF91 conferred robust protection against SVA over a contemporary period during primate evolution. We find that these new zinc fingers are important for binding the VNTR region of SVA, which in our experiments is necessary and sufficient for ZNF91-mediated repression. By extension of these and related findings, we propose that restriction of mobile element insertions in a mammalian genome is mediated in part by the presence of KZNFs evolved to recognize sequences within active TE families, and that the arms race created by this process has lead to the exceptional proliferation of lineage-specific ZNF genes in different mammalian lineages.

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