During his Ph.D in the laboratory of Professor Martin Allday, Dr. Kalchschmidt investigated the molecular mechanisms by which Epstein-Barr virus nuclear antigen 3C (EBNA3C) regulates host gene expression. Intrigued by the fact that EBNA3C can act as either activator or repressor of its target genes, he chose to study the most upregulated gene (AICDA) and the three most repressed genes (COBLL1, ADAM28, and ADAMDEC1). Changes to histone modifications at these loci correlated well with gene expression and indicated a two-step mechanism for EBNA3C-mediated repression. Surprisingly, recruitment of a Polycomb protein, generally linked to gene repression, was observed at all four genes, irrespective of being activated or repressed by EBNA3C. Furthermore, unexpected recruitment of the DNA-binding transcription factor RBPJ at EBNA3C binding sites was detected only when EBNA3C was functional. This challenged existing models of how RBPJ, a repressive transcription factor of the Notch signaling pathway, functions in EBNA3C-mediated gene regulation. Furthermore, the direct activation of AICDA by EBNA3C may play a role in EBV-associated lymphoma as it encodes a B cell-specific protein required for somatic hypermutation and class switch recombination, but also causes off-target DNA damage that can result in B cell lymphomagenesis.

To follow his interest and broaden his expertise in gene regulation, Dr. Kalchschmidt joined Dr. Rafael Casellas’ lab at the NIAMS to study the role of the mammalian Mediator complex in gene regulation. Mediator, a large 33 subunit multi-protein complex conserved across eukaryotes, plays an integral part in eukaryotic gene expression. In collaboration with the laboratory of Dr. Francisco Asturias, he worked on the genetic, functional, and structural characterization of the mammalian Mediator complex. CRISPR/Cas9 gene targeting identified essential subunits required for cell survival and a set of nonessential subunits that, upon deletion, mostly affected genes regulated by multiple enhancers. Structural analysis revealed that most essential subunits form a conserved core similar to yeast Mediator. Nonessential subunits, some of which are mammalian specific, added to the structural complexity of the mammalian Mediator. 

Contrary to current models that indicate Mediator acts as a topological bridge linking enhancers and promoters, depletion of Mediator does not alter genome architecture. Furthermore, conformational changes in Mediator were observed that increased interactions with RNA polymerase II. These results led to the proposal that the structural flexibility of Mediator enables it to integrate and relay regulatory signals between transcription factors and RNA polymerase II and that Mediator forms a functional, instead of an architectural, bridge linking enhancers and promoters.