Chromatin Structure and Function

Dr Gerrit koorsen – Chromatin structure and function

DNA in the eukaryotic cell nucleus is densely packaged within extensive nucleoprotein complexes known as chromatin.  The assembly not only facilitates the greater than 10,000-fold lengthwise compaction of DNA in order to accommodate large genomes (extended length ~ 2 m) within a nucleus with a comparatively small diameter (~ 10 mm), but also forms the substrate for all nuclear processes.  However, the compact state of a eukaryotic genome imposes considerable barriers to DNA access.  The histones, a class of highly conserved, basic proteins, constitute the main protein component of chromatin and directly establish and stabilize the structures that are responsible for the compaction of DNA through a hierarchical series of folding steps.   The nucleosome is the fundamental structural unit of chromatin and is composed of a histone octamer consisting of two copies of each of the core histones H2A, H2B, H3 and H4 around which approximately 146 bp of DNA is coiled (Luger et al., 1997).  It is now well established that core histone modifications (mostly occurring in their flexible N-terminal domains) constitute a complex “histone code” that defines the epigenetic state of chromatin domains through recruitment of effector molecules that direct distinct downstream events.  On average, a nucleosome is associated with one molecule of a fifth, linker histone (H1/H5) in vivo.  As a result of this association, an additional 20 bp of DNA (i.e. approximately 166 bp) is protected against digestion by micrococcal nuclease (MNase).  The resulting particle, which contains all five histones and approximately 166 bp of DNA is termed a "chromatosome".  Despite detailed structural knowledge of the nucleosome core particle, structural details of a chromatosome remain elusive.  However, it is clear that association of H1/H5 in a nucleosome results in the stable "sealing" of DNA exiting the nucleosome and that it facilitates the compaction of nucleosomes into the interphase chromatin fiber (the "30 nm-fiber").

The canonical linker histone consists of three domains, a central globular domain, flanked by basic, N- and C-terminal "tail" domains.  The globular domain, which specifically recognizes the nucleosome core, is highly conserved, whereas the tail domains exhibit pronounced sequence variation, suggesting a more diverse role of tail domains in different species and tissues.  Unlike the globular domain, the nature of the N-terminal domain of linker histone H1/H5 is a long-standing question.  However, since this domain is presumably located on the inside of the condensed chromatin fiber, its association with DNA and other histones is likely to impact on the structure of the chromatin fiber and fiber dynamics, which in turn affects most DNA-dependent processes and epigenetic regulation.  An understanding of the structure of the N-terminal domains of H1/H5 and its interaction with chromatin is therefore essential, not only to further our understanding of chromatin compaction, but also to understand the structural backdrop of epigenetic processes.  The main research focus of our group is to understand the structure and function of the N-terminal domains of several linker histone isotypes.

Apart from chromatin research, we are also engaged in interdisciplinary research together with the UJ Water and Health Research Unit and the UJ Optometry Department.  Details of specific projects currently underway are given below:

Identification of Vibrio cholera serotypes/biotypes by MALDI-TOF/MS

Co-investigator:   Dr T G Barnard

MSc Student:  Ronel Ferreira

Cholera, the highly epidemic diarrhoeal disease caused by Vibrio cholerae infection, continues to devastate many developing countries.  Due to the time-consuming methods for confirming Vibrio species, there is a definite need to improve detection methods for more rapid and sensitive detection.  Matrix-assisted Laser Desorption Ionization – Time-of-Flight (MALDI-TOF) mass spectrometry biotyping has recently emerged as a rapid and sensitive tool to identify cultured microorganisms.  We are investigating novel ways to adapt current MALDI-TOF/MS platforms for subspecies identification of South African Vibrio cholerae isolates.

 

Tear fluid composition of patients affected with dry eye disease and keratoconus

Co-investigator:  Prof. Wayne Gillan

MSc student: Rozanne Schnetler

Dry eye disease (DED) and keratoconus continue to affect the quality of life of many South Africans (and patients elsewhere) and in the case of keratoconus often leads to blindness.  However, details of the etiology of these diseases, their biochemical fingerprint as well as disease interactions remain uncertain.  We are taking a metabolomic approach to adressing these issues, and are specifically focusing our investigations on the lipid and immunological components of the precornial tear film.

 

From Left to Right: Ronél Ferreira; Dr Gerrit Koorsen; Rozanné Schnetler.