Gene expression is a tightly regulated system critical to defining cellular identity. Of the many regulatory components governing gene expression, this thesis looks at two specific components that can influence both transcriptional activation and repression.

First, I investigate the pleiotropic transcription factor (TF) Yin Yang 1 (YY1). Although this TF is developmentally essential and functions in many fundamental cell processes, there is still confusion regarding what contextual aspects dictate its function. I utilize protein biochemistry and biophysical assays to interrogate the interfaces responsible for RNA binding, a recently discovered capability of this TF. I reconcile previous studies claiming that different regions of the protein confer these nucleic acid interactions by demonstrating that there are multiple domains of YY1 that can bind RNA. My work also uncovers a previously unannotated intramolecular inhibitory mechanism that YY1’s N-terminus can impose upon its zinc finger module.

Second, I look into the nucleosomal-existence of activating (H3K4me3) and repressive (H3K27me3) chromatin marks, in the context of the bivalency model. An entire field of chromatin biology has canonized bivalent chromatin as a functional co-occurence essential for cellular differentiation. In this thesis I address specific pitfalls of the conventional way that chromatin immunoprecipitation (ChIP) is performed and present methodologies developed by the Ruthenburg lab (ICeChIP and ReICeChIP) that rigorously address these drawbacks and provide quantitative insight to the distribution of these chromatin marks. With these observations, we directly call the bivalency model into question as we assess the chromatin states of mouse embryonic stem cells through differentiation to neuronal precursor cells.

Maintaining the balance between gene activation and repression requires multiple layers of regulation. This work provides frameworks for deeper investigations of gene regulation in the future.