PRDM proteins: important players in differentiation and disease

The PRDM family has recently spawned considerable interest as it has been implicated in fundamental aspects of cellular differentiation and exhibits expanding ties to human diseases. The PRDMs belong to the SET domain family of histone methyltransferases, however, enzymatic activity has been determined for only few PRDMs suggesting that they act by recruiting co-factors or, more speculatively, confer methylation of non-histone targets. Several PRDM family members are deregulated in human diseases, most prominently in hematological malignancies and solid cancers, where they can act as both tumor suppressors or drivers of oncogenic processes. The molecular mechanisms have been delineated for only few PRDMs and little is known about functional redundancy within the family. Future studies should identify target genes of PRDM proteins and the protein complexes in which PRDM proteins reside to provide a more comprehensive understanding of the biological and biochemical functions of this important protein family.     

HMG chromosomal proteins in development and disease

The high mobility group (HMG) proteins are a superfamily of abundant and ubiquitous nuclear proteins that bind to DNA and nucleosomes and induce structural changes in the chromatin fiber. They are important in chromatin dynamics and influence DNA processing in the context of chromatin. Results emerging from studies of human disease, genetically modified mice and cells with altered HMG expression indicate that the expression of the HMG proteins is developmentally regulated and that changes in HMG protein levels alter the cellular phenotype and can lead to developmental abnormalities and disease. Here, we focus on the biological function of HMG proteins and highlight their possible roles in cellular differentiation and in the etiology of various diseases.     

Nonhistone proteins HMG1 and HMG2 unwind DNA double helix.

In a previous communication we have shown that both HMG1 and HMG2 nonhistone proteins change the DNA helical structure and the binding of HMG1 and HMG2 to DNA induces a net unwinding equivalent of DNA double helix (Javaherian, K., Liu, L. F. and Wang, J. C. (1978) Science, 199, 1345-1346). Employing melting absorption technique, we now show that in the presence of salt HMG1 and HMG2 destabilize DNA whereas in the absence of salt, they both stabilize DNA molecules. Consequently the folded structure of HMG must play an important role in melting DNA. Furthermore, by measuring topological winding number using competition unwinding experiments, we conclude that HMG1 has a higher affinity for a single-stranded DNA relative to double-stranded DNA. These results together suggest that HMG1 and HMG2 unwind DNA double helix by local denaturation of the DNA base pairs. The net unwinding angles have been measured to be 22 degrees and 26 degrees per molecule of HMG1 and HMG2 respectively.     

Mathematical approaches to differentiation and gene regulation

We consider some mathematical issues raised by the modelling of gene networks. The expression of genes is governed by a complex set of regulations, which is often described symbolically by interaction graphs. These are finite oriented graphs where vertices are the genes involved in the biological system of interest and arrows describe their interactions: a positive (resp. negative) arrow from a gene to another represents an activation (resp. inhibition) of the expression of the latter gene by some product of the former. Once such an interaction graph has been established, there remains the difficult task to decide which dynamical properties of the gene network can be inferred from it, in the absence of precise quantitative data about their regulation. There mathematical tools, among others, can be of some help. In this paper we discuss a rule proposed by Thomas according to which the possibility for the network to have several stationary states implies the existence of a positive circuit in the corresponding interaction graph. We prove that, when properly formulated in rigorous terms, this rule becomes a theorem valid for several different types of formal models of gene networks. This result is already known for models of differential [C. Soulé, Graphic requirements for multistationarity, ComPlexUs 1 (2003) 123-133] or Boolean [E. Rémy, P. Ruet, D. Thieffry, Graphic requirements for multistability and attractive cycles in a boolean dynamical framework, 2005, Preprint] type. We show here that a stronger version of it holds in the differential setup when the decay of protein concentrations is taken into account. This allows us to verify also the validity of Thomas' rule in the context of piecewise-linear models. We then discuss open problems.     

Differential binding of chromosomal proteins HMG1 and HMG2 to superhelical DNA

The binding of chromosomal proteins HMG1 and HMG2 to various DNA structures was examined by a nitrocellulose filter binding assay using a 32P labelled supercoiled plasmid. Binding assays and competition experiments indicated that HMG2 has a higher affinity than HMG1 for supercoiled DNA. Studies at various ionic strengths and pH values reveal differences in the interaction of the two proteins with DNA. The results suggest that HMG1 and HMG2 are involved in distinguishable cellular functions.     

Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.     

On the presence of the chromosomal proteins HMG I and HMG Y in rat organs

Using antiserum raised against HMG I, we have shown that HMG I and HMG Y are present in perchloric acid extracts of kidney, lung, heart, brain, liver and intestine in the rat, suggesting that the expression of these proteins may not be dependent upon proliferative activity. The results also show that the ratio between HMG I and HMG Y varies between different organs.     

Immunochemical studies of high mobility group non-histone chromatin proteins HMG 1 and HMG 2

Sera were raised to non-histone chromatin proteins HMG 1 and HMG 2. Immunoperoxidase staining localised these proteins on chromosomes during mitosis and indicated a cell cycle-related variation in these proteins during interphase. Some species differences in HMG 1 and HMG 2 were also observed.     

On the theories of gene regulation and differentiation in eukaryotes

The interrelationships among recent theories on the regulation of gene activity and differentiation in higher organisms are reviewed. Interpretations within these theories of the various components of chromosomes are re-evaluated and a unified conceptual framework of hierarchical genetic control mechanisms in eukaryotes is presented.