ORC2 regulation of human gene expression shows unexpected breadth and scale

BIRMINGHAM, Ala. – Origin-recognition complex, or ORC, plays an unexpectedly broad role in the regulation of human cell gene expression, according to a study in the journal Cell Reports.

“This is the first detailed study of how and where ORC regulates epigenetics and gene expression in human cells,” said Anindya Dutta, Ph.D., leader of the study and chair of the University of Alabama at Birmingham Department of Genetics. “The unanticipated scale and breadth of the regulation opens new chapters in ORC biology.”

The six-subunit complex was discovered in yeast as essential to begin DNA replication. However, a variety of experiments, particularly in lower eukaryotic cells like yeast, suggested a broader role for ORC in regulating the structure of chromatin, the DNA-protein material that contains all the genes of the cell. Chromatin is closely packed into each cell nucleus, and changes in DNA structure — both relaxing or condensing the chromatin and adding or removing epigenetic marks on DNA or histone proteins — are part of the complex cell mechanisms that turn genes on or off.

The researchers previously created three knockdown mutations of ORC subunits in human cancer cell lines. The current study reports that individual subunits, rather than the entire ORC complex, are able to bind many unique DNA sites.

“Although the ORC subunits should bind DNA as part of a common six-subunit ORC, there are thousands of sites in the genome where one subunit binds but not another,” Dutta said.

Importantly, those unique binding sites independently affected gene expression. The ORC2 subunit at some binding sites, as expected from studies with lower cells, compacted the chromatin and attracted repressive histone marks, both of which make genes less accessible for expression. But surprisingly, say the UAB Marnix E. Heersink School of Medicine and University of Virginia School of Medicine researchers, ORC2 binding at many other sites activated chromatin for gene expression.

Furthermore, ORC2 bound to DNA also prevented the acquisition of CTCF at some focal sites in the genome. CTCF, called a “master weaver” of the genome, controls the formation of DNA loops and how DNA is folded. This, in turn, affects gene expression.

“Our results suggest that ORC2’s binding to the chromatin appears to keep CTCF away from certain sites, and this could be because of the local repression of chromatin at those sites,” Dutta said. “In the absence of ORC2, CTCF binds to these sites and creates new anchor points for loop formation.”

At two gene loci the researchers studied, increased looping in the absence of ORC2 isolated a known gene enhancer from the gene promoter, the DNA sequence where mRNA synthesis begins. This looping, researchers found, depressed mRNA expression and allowed spread of repressive epigenetic marks.

The cell lines used in the study have knockdowns of the ORC1, ORC2 or ORC5 subunits. Although they grow more slowly than wildtype cells, they survive, proliferate and replicate DNA with the normal complement of origins of replication. Analysis of these knockdown mutant cell lines and “rescue” cell lines where the wildtype genes for ORC1, ORC2 or ORC5 were added back to the mutants let researchers carefully examine ORC’s role in epigenetics, gene expression and higher-order chromosome structure, yielding a wealth of information about how ORC subunits regulate gene expression in mammalian cells.

One challenge to studying DNA structure in the human cell is the immense size of the genome. A single cell’s DNA — if connected end-to-end and pulled taught like a wire — would stretch 6 and a half feet. This long polynucleotide strand of DNA undergoes dynamic structural changes inside the nucleus. Some segments wind tightly around proteins to compact the DNA, while other segments loosen to allow access to genes.

The Cell Reports study, “Regulation of epigenetics and chromosome structure by human ORC2,” was led corresponding authors Dutta, Zhangli Su, Ph.D., UAB Department of Genetics, and Chongzhi Zang, Ph.D., University of Virginia School of Medicine.

Co-authors are Mengxue Tian and Zhenjia Wang, University of Virginia School of Medicine, Charlottesville, Virginia; Etsuko Shibata and Yoshiyuki Shibata, UAB Department of Genetics; and Tianyi Yang and Fulai Jin, Case Western Reserve University, Cleveland, Ohio.

UAB Genetics is a department in the Marnix E. Heersink School of Medicine.