Satb1 as Traffic Cop: Directing Chromatin Compaction, Transcription, and Molecular Flux

Satb1 as Traffic Cop:/nDirecting Chromatin Compaction, Transcription, and Molecular Flux/nby/nBrett Jeffrey Schofield/nDoctor of Philosophy in Molecular and Cell Biology/nUniversity of California, Berkeley/nProfessor Jan Liphardt, Co-chair/nProfessor Carlos Bustamante, Co-chair/nThe ability to carefully regulate one's genome in response to environmental cues is a fundamental requirement for life. Research into how cells accomplish this has been vigorously pursued ever since the one-gene one-protein hypothesis was introduced in 1941. And although a wealth of information has been garnered from this work, the field continually finds new and unexpected mechanisms that cells utilize to regulate transcription. The most recent discovery being that the location and organization of individual chromatin strands can have a significant impact on gene expression. However, the consequences of specific chromatin configurations and the mechanisms that generate them are only beginning to be understood./nIn this dissertation, I explore the capabilities and function of one chromatin architectural protein - the Special AT-rich sequence Binding protein 1 (Satb1). I was initially interested in characterizing several basic properties of Satb1 for which there are conflicting or incomplete data typically based on in vitro experiments. Using a variety of in vivo techniques, I demonstrated that Satb1 forms stable and long-lived homodimers whose formation is dependent on the N-terminal PDZ domain, but independent of DNA binding. I have also shown that the cooperation between the PDZ domain and either the Cut1 or Cut2 DNA binding domains is essential for high-affinity DNA binding in vivo. The removal of the Homeo domain, however, has little impact on the binding kinetics of Satb1. Interestingly, the diffusion of Satb1 is slowest in a 4-500nm wide region at the interface of high and low density DNA, indicating the presence of a large number of high-affinity Satb1 binding sites in this zone./nThe competition between Satb1 and its close homologue Satb2 has been shown to beimportant in determining Embryonic Stem (ES) cell fate, and may play an important role in transitioning from embryonic (ϵ) to fetal (Gγ, Aγ) globin production during erythrocyte development. However, the mechanism behind this competition is unknown. My experiments indicate that these proteins are able to effectively compete at the level of DNA binding, although some high-affinity sites maintain a preference for one protein over the other. Furthermore, they can form heterodimers, raising the possibility that the unique protein-interacting interface offered by the heterodimer recruits a different pool of partners than either homodimer./nFinally, I examined what changes Satb1 makes to local and global chromatin architecture, and how they differ from those caused by Satb2, or the insulator element protein CTCF. When synthetically targeted to a trans-gene array by fusion with the lac repressor (LacR), the MAR binding proteins Satb1 and Satb2 have a bidirectional effect on local chromatin compaction. They have the ability to open heterochromatic arrays, and condense euchromatic arrays. The final array configuration is independent of its initial state. The difference between the proteins is the final size of the array - targeting of Satb1 results in a more compact array than does Satb2. CTCF, in contrast, as a uni-directional effect. It significantly decompacts heterochromatic arrays, but has no effect on euchromatic ones. Single molecule RNA Fluorescence In Situ Hybridization (FISH) shows that these dramatic alterations to chromatin architecture are mirrored in the expression of the underlying genes. Satb1-bound arrays produce an intermediate number of transcripts whose number is independent of the initial transcript number. Likewise, CTCF significantly increases the number of transcripts produced from a heterochromatic array, but has little effect on the number produced by a euchromatic array./nSatb1 has a unique role in determining the diffusive route of inert molecules like EGFP in the nucleus. Such molecules preferentially diffuse into areas of similar DNA density. Transits between areas of vastly different chromatin density are infrequent and occur in bidirectional bursts. Expression of Satb1, but not CTCF, increases the frequency of these molecular transits, allowing proteins like EGFP greater access to high DNA density regions. Satb1 is the only protein identified so far to alter the strength of this diffusive barrier.

Advisor: Liphardt, Jan
Bustamante, Carlos