DNA Bending by the Mammalian High-Mobility Group Protein AT Hook 2

Abstract

The mammalian high mobility group protein AT hook 2 (HMGA2) is a DNA binding protein that specifically recognizes the minor groove of AT-rich DNA sequences. Disruption of its expression pattern is directly linked to oncogenesis and obesity. In this paper, we constructed a new plasmid pBendAT to study HMGA2-induced DNA bending. pBendAT carries a 230 bp DNA segment containing 5 pairs of restriction enzyme sites, which can be used to produce a set of DNA fragments of identical length to study protein-induced DNA bending. The DNA fragments of identical length can also be generated using PCR amplification. Since pBendAT does not contain more than 3 consecutive AT base pairs, it is suitable for the determination of DNA bending induced by proteins recognizing AT-rich DNA sequences. Indeed, using pBendAT, we demonstrated that HMGA2 is a DNA bending protein and bends all three tested DNA binding sequences of HMGA2, SELEX1, SELEX2, and PRDII. The DNA bending angles were estimated to be 34.2, 33.5, and 35.4°, respectively. Keywords HMGA2; AT hook; DNA bendingThe mammalian high mobility group protein AT-hook 2 (HMGA2) is a transcriptional factor involved in mesenchymal cell differentiation and transformation (1-3). It plays an important role in fat cell proliferation (2) and is considered to be a potential target for treatment of obesity (2,4). HMGA2 is also an onco-protein associated with the formation of a variety of tumors including benign tumors, such as uterine leiomyomas (5), lipomas (6,7) and pulmonary chondroid hamartomas (8), and malignant tumors, such as lung cancers (9-11), breast cancers (12), and leukemia (13). Interestingly, the expression level of HMGA2 was demonstrated to be correlated with the degree of malignancy, the existence of metastasis, and a poor prognosis for certain cancers (14,15). These results suggest that HMGA2 is a potential target for treatment of these cancers (4,16,17).HMGA2 is a DNA binding protein containing three "AT hook" DNA binding domains that specifically recognize the minor groove of AT rich DNA sequences (18). Although each "AT hook" DNA binding domain binds to five AT base pairs (18,19), the high-affinity binding of HMGA2 requires two to three appropriated spaced 5-bp AT-rich DNA sequences as a single Here we decided to study HMGA2-induced DNA conformational change, specifically DNA bending. Since each "AT hook" DNA binding domain contains 5 to 6 positively charges, it should bend the DNA locus upon binding to the AT-rich DNA sequences (22). The challenge is to find a short DNA fragment that does not contain 4 to 5 consecutive AT base pairs for the gel permutation assay. In this case, we can clone one HMGA2 binding site into this short DNA fragment and precisely determine the HMGA2-induced DNA-bending angle without the interference caused by HMGA2 binding to other AT-rich sequences, i.e. sequences containing more than 3 consecutive AT base pairs. In this study, we constructed a new plasmid pBendAT to study HMGA2-induced DNA bending. pBendAT carries a 230 bp DNA sequence ...

High mobility group (HMG) proteins: Modulators of chromatin structure and DNA repair in mammalian cells

“…Negative regulation, on the other hand, is thought to involve interference with enhanceosome formation as a consequence of HMGA binding that prevents transcription factors from associating with their DNA recognition sequences on promoters [85,90]. The fact that HMGA proteins can bend, straighten, unwind and supercoil DNA substrates in vitro without requiring energy (i.e., ATP) input [108][109][110][111] likely contributes to their ability to bind to, and induce structural changes in, gene promoters during transcriptional regulation in vivo.…”

The Role of Hmga2 in the Proliferation and Expansion of a Hematopoietic Cell in Myeloproliferative Neoplasms

“…DNA -binding AT -hook domains of HMGA2, which are encoded by first three exons of the HMGA2 gene, can modulate transcription by affecting the DNA conformation of specific AT -rich regulatory elements promoting transcriptional activity 60,61) . The HMGA2 protein is important in a wide spectrum of biological processes, including cell proliferation, cell -cycle progression, apoptosis, and senescence 62,63) .…”

Molecular Basis for Sequence‐Dependent Induced DNA Bending

“…Therefore the malleability of DNA impacts recognition specificity and has direct functional benefit. [3,5,7,11] AT sequences of the same composition have two limiting sequence arrangements, either A-tracts, with all A bases on one strand and all T on the other, or alternating A and T bases on both strands. Extensive experimental evidence indicates that these two sequence arrangements have quite different structures and properties.…”