Raymond Reeves

High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function.

Publisher Summary This chapter summarizes recent information on the function of high-mobility-group (HMG) proteins. Advances in this field were made primarily by elucidating the structure of these proteins and by understanding their mode of interactions with DNA and chromatin. Renewed interest in these proteins is because of the finding that the DNA-binding domains of many regulatory proteins share common elements with the HMG-1/-2 chromosomal protein family. The HMG proteins are among the largest and best characterized group of nonhistone chromosomal proteins. Members of this protein group are found in all the cells of higher eukaryotes. Most of the data suggest that HMG proteins are associated with selected regions in chromatin and this association affects the architecture and increases the structural complexity of the chromatin fiber. The human HMG-I(Y) gene is located on the short arm of chromosome 6 in a region involved in rearrangements, translocations, and other abnormalities correlated with a number of human cancers.

The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif.

The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51–90), and a DNA dodecamer containing the PRDII site of the interferon-β promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contacts. The absence of one of these modules in the third DNA binding domain accounts for its ∼100 fold reduced affinity relative to the second one.

Transcriptionally active chromatin

Eukaryotic chromatin has a dynamic, complex hierarchical structure. Active gene transcription takes place on only a small proportion of it at a time. While many workers have tried to characterize active chromatin, we are still far from understanding all the biochemical, morphological and compositional features that distinguish it from inactive nuclear material. Active genes are apparently packaged in an altered nucleosome structure and are associated with domains of chromatin that are less condensed or more open than inactive domains. Active genes are more sensitive to nuclease digestions and probably contain specific nonhistone proteins which may establish and/or maintain the active state. Variant or modified histones as well as altered configurations or modifications of the DNA itself may likewise be involved. Practically nothing is known about the mechanisms that control these nuclear characteristics. However, controlled accessibility to regions of chromatin and specific sequences of DNA may be one of the primary regulatory mechanisms by which higher cells establish potentially active chromatin domains. Another control mechanism may be compartmentalization of active chromatin to certain regions within the nucleus, perhaps to the nuclear matrix. Topological constraints and DNA supercoiling may influence the active regions of chromatin and be involved in eukaryotic genomic functions. Further, the chromatin structure of various DNA regulatory sequences, such as promoters, terminators and enhancers, appears to partially regulate transcriptional activity.

Architectural transcription factor HMGI(Y) promotes tumor progression and mesenchymal transition of human epithelial cells.

ABSTRACT Numerous studies have demonstrated that overexpression or aberrant expression of the HMGI(Y) family of architectural transcription factors is frequently associated with both neoplastic transformation of cells and metastatic tumor progression. Little is known, however, about the molecular roles played by the HMGI(Y) proteins in these events. Here we report that human breast epithelial cells harboring tetracycline-regulated HMGI(Y) transgenes acquire the ability to form both primary and metastatic tumors in nude mice only when the transgenes are actively expressed. Unexpectedly, the HMG-Y, rather than the HMG-I, isoform of these proteins is the most effective elicitor of both neoplastic transformation and metastatic progression in vivo. Furthermore, expression of either antisense or dominant-negative HMGI(Y) constructs inhibits both the rate of proliferation of tumor cells and their ability to grow anchorage independently in soft agar. Array analysis of transcription profiles demonstrates that the HMG-I and HMG-Y isoform proteins each modulate the expression of distinctive constellations of genes known to be involved in signal transduction, cell proliferation, tumor initiation, invasion, migration, induction of angiogenesis, and colonization. Immunohistochemical analyses of tumors formed in nude mice indicate that many have undergone an epithelial-mesenchymal transition in vivo. Together, these findings demonstrate that overexpression of the HMGI(Y) proteins, more specifically, the HMG-Y isoform protein, is causally associated with both neoplastic transformation and metastatic progression and suggest that induction of integrins and their signaling pathways may play significant molecular roles in these biological events.

Network of Dynamic Interactions between Histone H1 and High-Mobility-Group Proteins in Chromatin

ABSTRACT Histone H1 and the high-mobility group (HMG) proteins are chromatin binding proteins that regulate gene expression by modulating the compactness of the chromatin fiber and affecting the ability of regulatory factors to access their nucleosomal targets. Histone H1 stabilizes the higher-order chromatin structure and decreases nucleosomal access, while the HMG proteins decrease the compactness of the chromatin fiber and enhance the accessibility of chromatin targets to regulatory factors. Here we show that in living cells, each of the three families of HMG proteins weakens the binding of H1 to nucleosomes by dynamically competing for chromatin binding sites. The HMG families weaken H1 binding synergistically and do not compete among each other, suggesting that they affect distinct H1 binding sites. We suggest that a network of dynamic and competitive interactions involving HMG proteins and H1, and perhaps other structural proteins, constantly modulates nucleosome accessibility and the local structure of the chromatin fiber.

Nuclear functions of the HMG proteins

Although the three families of mammalian HMG proteins (HMGA, HMGB and HMGN) participate in many of the same nuclear processes, each family plays its own unique role in modulating chromatin structure and regulating genomic function. This review focuses on the similarities and differences in the mechanisms by which the different HMG families impact chromatin structure and influence cellular phenotype. The biological implications of having three architectural transcription factor families with complementary, but partially overlapping, nuclear functions are discussed.

High Mobility Group Protein I(Y) Is Required for Function and for c-Rel Binding to CD28 Response Elements within the GM-CSF and IL-2 Promoters

CD28 response elements (CD28REs) within cytokine promoters are variant NF-kappaB-binding sites and are essential for transcription in response to CD28 receptor activation in T cells. We show that the CK-1 element (CD28RE) within the GM-CSF promoter binds the RelA and c-Rel transcription factors in response to CD28 activation. We further show that the high mobility group protein HMG I(Y) can bind to the CD28REs of both GM-CSF and IL-2 and that this binding is critical for c-Rel, but not RelA, binding. A second NF-kappaB site in the GM-CSF promoter that binds p50 and RelA, but neither c-Rel nor HMG I(Y), failed to respond to CD28 activation. Expression of HMG I or c-Rel antisense RNA inhibited CD28 activation of the IL-2 and GM-CSF promoters, implying that HMG I(Y) enhancement of c-Rel binding plays an important role in the activity of the CD28REs.

The Role of High-Mobility Group I(Y) Proteins in Expression of IL-2 and T Cell Proliferation

The high-mobility group I(Y) (HMGI(Y)) family of proteins plays an important architectural role in chromatin and have been implicated in the control of inducible gene expression. We have previously shown that expression of HMGI antisense RNA in Jurkat T cells inhibits the activity of the IL-2 promoter. Here we have investigated the role of HMGI(Y) in controlling IL-2 promoter-reporter constructs as well as the endogenous IL-2 gene in both Jurkat T cells and human PBL. We found that the IL-2 promoter has numerous binding sites for HMGI(Y), which overlap or are adjacent to the known transcription factor binding sites. HMGI(Y) modulates binding to the IL-2 promoter of at least three transcription factor families, AP-1, NF-AT and NF-κB. By using a mutant HMGI that cannot bind to DNA but can still interact with the transcription factors, we found that DNA binding by HMGI was not essential for the promotion of transcription factor binding. However, the non-DNA binding mutant acts as a dominant negative protein in transfection assays, suggesting that the formation of functional HMGI(Y)-containing complexes requires DNA binding as well as protein:protein interactions. The alteration of HMGI(Y) levels affects IL-2 promoter activity not only in Jurkat T cells but also in PBL. Importantly, we also show here that expression of the endogenous IL-2 gene as well as proliferation of PBL are affected by changes in HMGI(Y) levels. These results demonstrate a major role for HMGI(Y) in IL-2 expression and hence T cell proliferation.

Recruitment of SWI/SNF to the Human Immunodeficiency Virus Type 1 Promoter

ABSTRACT Following human immunodeficiency virus type 1 (HIV-1) integration into the host cells genome, the 5′ long terminal repeat (LTR) is packaged into a highly specific chromatin structure comprised of an array of nucleosomes positioned with respect to important DNA sequence elements that regulate the transcriptional activity of the provirus. While several host cell factors have been shown to be important for chromatin remodeling and/or basal transcription, no specific mechanism that relieves the transcriptional repression imposed by nuc-1, a positioned nucleosome that impedes the start site of transcription, has been found. Since phorbol esters cause the rapid disruption of nuc-1 and markedly stimulate HIV-1 transcription, we looked for protein factors that associate with this region of the HIV-1 promoter in a phorbol-ester-dependent manner. We report here that ATF-3, JunB, and BRG-1 (the ATPase subunit of the 2-MDa human chromatin remodeling machine SWI/SNF) are recruited to the 3′ boundary of nuc-1 following phorbol myristate acetate stimulation in Jurkat T cells. Analysis of the recruitment of BRG-1 in nuclear extracts prepared from Jurkat T cells and reconstitution of an in vitro system with purified components demonstrate that ATF-3 is responsible for targeting human SWI/SNF (hSWI/SNF) to the HIV-1 promoter. Importantly, this recruitment of hSWI/SNF required HMGA1 proteins. Further support for this conclusion comes from immunoprecipitation experiments showing that BRG-1 and ATF-3 can exist together in the same complex. Although ATF-3 clearly plays a role in the specific targeting of BRG-1 to the HIV-1 promoter, the maintenance of a stable association between BRG-1 and chromatin appears to be dependent upon histone acetylation. By adding BRG-1 back into a BRG-1-deficient cell line (C33A cells), we demonstrate that trichostatin A strongly induces the 5′-LTR-driven reporter transcription in a manner that is dependent upon BRG-1 recruitment.

Why target PIM1 for cancer diagnosis and treatment

The highly conserved proto-oncogenic protein PIM1 is an unusual serine or threonine kinase, in part because it is constitutively active. Overexpression of PIM1 experimentally leads to tumor formation in mice, while complete knockout of the protein has no observable phenotype. It appears to contribute to cancer development in three major ways when it is overexpressed; by inhibiting apoptosis, by promoting cell proliferation and by promoting genomic instability. Expression in normal tissues is nearly undetectable. However, in hematopoietic malignancies and in a variety of solid tumors, increased PIM1 expression has been shown to correlate with the stage of disease. This characteristic suggests it can serve as a useful biomarker for cancer diagnosis and prognosis. Several specific and potent inhibitors of PIM1’s kinase activity have also been shown to induce apoptotic death of cancer cells, to sensitize cancer cells to chemotherapy and to synergize with other anti-tumor agents, thus making it an attractive therapeutic target.