Chromatin Function: A Network of Competitive Interactions Between Nucleosome Binding Proteins

Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, and Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol 24: 4321–8, 2004.

he ability of regulatory factors to access their binding sites in nucleosomes affects DNA-related processes in chromatin, such as transcription, replication, repair, and genetic recombination. These processes are modulated by nuclear proteins, such as the linker histone H1 and members of the high mobility group (HMG) families, which reduce or enhance the access to nucleosomes, respectively. Cells from higher eukaryotes contain about eight H1 variants in amounts sufficient to bind to over 80% of the nucleosomes. HMGs are nuclear proteins that interact with nucleosomes and change the “architecture” of chromatin. The HMGs are subdivided into three families, HMGA, HMGB, and HMGN, each with a distinct structural motif through which it binds to chromatin. An interplay between H1 and HMGs, which have opposite effects on chromatin compaction, could be part of the mechanism that imparts flexibility to chromatin fiber and could play an important role in determining the cellular phenotype.

Together with Dr. Tom Misteli of the Laboratory of Receptor Biology and Gene Expression, who developed approaches to study the chromatin binding of proteins in living cells, we are investigating whether chromatin binding proteins compete for nucleosome binding sites. In living cells, chromatin binding proteins such as H1 and HMG proteins are in constant motion and interact only transiently with their chromatin binding sites. The proteins bind to chromatin in a “stop-and-go” process, the “stop” being the stage in which they are bound and the “go” being the stage in which the protein moves from one binding site to another. The length of the “stop” and the “go” steps depends on the binding constant of the protein for a particular site. Their sum determines the overall apparent mobility of the protein in the nucleus. For example, a protein that binds strongly to chromatin will display a low mobility, as the binding step will slow down its movement. The mobility of proteins in living cells is experimentally monitored by techniques such as fluorescence recovery after photobleaching (FRAP).

To test for competition between H1 and HMG chromatin binding proteins, we microinjected individual HMG proteins (HMGA, HMGB, or HMGN) into cells expressing fluorescent H1, and compared the mobility of H1 in injected cells versus non-injected cells. We reasoned that if HMG and H1 compete with each other for chromatin sites, an increase in the intracellular concentration of HMG should decrease the binding of H1. Indeed, for all three HMG protein families, the mobility of fluorescent H1 was higher in injected cells than in control cells. Thus, each HMG protein weakens the binding of H1 to chromatin. The inhibition is specific since HMG mutant proteins, which do not bind to chromatin, did not affect the binding of H1 to chromatin. HMG proteins decreased the chromatin binding of H1 in a dose-dependent fashion, a typical feature of molecular competition. These results indicate that HMGs and H1 proteins compete with each other for chromatin binding sites. Because all members of the three HMG families bind to nucleosomes and compete with H1, we next tested whether the HMGs compete with each other. While competitions were observed between HMG proteins of the same family (e.g., between HMGN1 and HMGN2), no competition could be observed between HMGs from different families (i.e., HMGB with HMGN). Thus, there is a certain level of specificity in the competition. Because HMGs do not compete with each other, we thought that they might work together to reduce H1 binding. Thus, we mixed different HMGs together and monitored the impact of their injection, individually or mixed, on H1 binding to chromatin. We found that the effect of HMG mixes on H1 binding is stronger than the sum of the effects of each HMG injected separately. Thus, the HMG proteins compete with H1 synergistically.

Our studies indicate that members of four families of structural proteins that regulate chromatin structure and activity form a network of competitive interactions on nucleosomes. It is likely that such competitive interactions occur between other classes of chromatin binding factors. The realization that chromatin binding proteins function within a network has important ramifications for understanding mechanisms that regulate the cellular response to biological signals. Conceivably, the cell can tailor a specific response by adjusting the equilibrium of the binding and activity of the various members of a protein network. Thus, the dynamic competition between chromatin binding proteins could affect numerous DNA-related processes and perhaps play a role in the epigenetic regulation of gene expression.

Frederic Catez, PhD
Visiting Fellow
catezf@mail.nih.gov

Michael Bustin, PhD
Principal Investigator
Laboratory of Metabolism
NCI-Bethesda, Bldg. 37/Rm. 3122A
Tel: 301-496-5234
Fax: 301-496-8419
bustin@helix.nih.gov