The behavior of SATB1, a MAR-binding protein, in response to apoptosis stimulation

As a MAR-binding protein, SATB1 regulates genes by folding chromatin into a loop domain. Apoptosis is known to be accompanied by a collapse of nuclear architecture and cleavage of condensing chromatin into oligonucleosomal fragments. To further understand the functional role of MAR-binding proteins during apoptosis we investigated the relationship of the behavior of SATB1 and the collapse of nuclear architecture in Jurkat cells with immunostaining and Western blot analysis. We demonstrated that SATB1 formed special three-dimensional network distributions during early apoptosis. The distribution change of SATB1 was associated with cleavage of the protein and accompanied by the nuclear architecture collapse. Cleavage of SATB1 was mediated by caspase-3 and was apoptosis specific. Our observations further support the notion that early proteolysis of MAR-binding proteins might represent a universal mechanism that renders these DNA sites vulnerable to endonucleolysis.     

The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1–1.1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island–containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear “halos” with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type–specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell–specific gene regulation.     

Structural basis for recognition of the matrix attachment region of DNA by transcription factor SATB1

Special AT-rich sequence binding protein 1 (SATB1) regulates gene expression essential in immune T-cell maturation and switching of fetal globin species, by binding to matrix attachment regions (MARs) of DNA and inducing a local chromatin remodeling. Previously we have revealed a five-helix structure of the N-terminal CUT domain, which is essentially the folded region in the MAR-binding domain, of human SATB1 by NMR. Here we determined crystal structure of the complex of the CUT domain and a MAR DNA, in which the third helix of the CUT domain deeply enters the major groove of DNA in the B-form. Bases of 5'-CTAATA-3' sequence are contacted by this helix, through direct and water-mediated hydrogen bonds and apolar and van der Waals contacts. Mutations at conserved base-contacting residues, Gln402 and Gly403, reduced the DNA-binding activity, which confirmed the importance of the observed interactions involving these residues. A significant number of equivalent contacts are observed also for typically four-helix POU-specific domains of POU-homologous proteins, indicating that these domains share a common framework of the DNA-binding mode, recognizing partially similar DNA sequences.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Crystal Structure of the Ubiquitin-like Domain-CUT Repeat-like Tandem of Special AT-rich Sequence Binding Protein 1 (SATB1) Reveals a Coordinating DNA-binding Mechanism

SATB1 is essential for T-cell development and growth and metastasis of multitype tumors and acts as a global chromatin organizer and gene expression regulator. The DNA binding ability of SATB1 plays vital roles in its various biological functions. We report the crystal structure of the N-terminal module of SATB1. Interestingly, this module contains a ubiquitin-like domain (ULD) and a CUT repeat-like (CUTL) domain (ULD-CUTL tandem). Detailed biochemical experiments indicate that the N terminus of SATB1 (residues 1–248, SATB1^(1–248)), including the extreme 70 N-terminal amino acids, and the ULD-CUTL tandem bind specifically to DNA targets. Our results show that the DNA binding ability of full-length SATB1 requires the contribution of the CUTL domain, as well as the CUT1-CUT2 tandem domain and the homeodomain. These findings may reveal a multiple-domain-coordinated mechanism whereby SATB1 recognizes DNA targets.     

The structural basis for the oligomerization of the N-terminal domain of SATB1

Special AT-rich sequence-binding protein 1 (SATB1) is a global chromatin organizer and gene expression regulator essential for T-cell development and breast cancer tumor growth and metastasis. The oligomerization of the N-terminal domain of SATB1 is critical for its biological function. We determined the crystal structure of the N-terminal domain of SATB1. Surprisingly, this domain resembles a ubiquitin domain instead of the previously proposed PDZ domain. Our results also reveal that SATB1 can form a tetramer through its N-terminal domain. The tetramerization of SATB1 plays an essential role in its binding to highly specialized DNA sequences. Furthermore, isothermal titration calorimetry results indicate that the SATB1 tetramer can bind simultaneously to two DNA targets. Based on these results, we propose a molecular model whereby SATB1 regulates the expression of multiple genes both locally and at a distance.     

SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes

Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3’-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.     

一种宏观基因调控分子SATB1

自身多聚化的SATB1(special AT-rich sequences binding protein 1)围绕异染色质形成笼状结构分布在细胞核中,SATB1不仅结合染色质DNA的核基质结合区(matrix attachment regions,MARs),也结合核基质,能够使DNA锚定在核基质并形成袢环状结构(loop)。SATB1的磷酸化、乙酰化和小泛素化样修饰可调节其DNA结合能力和细胞核内亚结构的定位;SATB1与多种蛋白质相互作用,能够募集染色质重塑复合物和组蛋白修饰酶,实现对其靶基因表达的时空特异性调控。SATB1在调节细胞分化、细胞凋亡、肿瘤生长与转移和X染色体失活等方面起到重要作用,并有可能成为肿瘤转移的治疗靶点。     

The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.     

Characterization of apoptotic pathway associated with caspase-independent excision of DNA loop domains

Excision of chromatin loop domains and internucleosomal DNA fragmentation are widely considered as consecutive stages of chromatin disassembly during apoptosis. We report here on apoptosis induced by staurosporine in NB-2a neuroblastoma cells, which was accompanied by excision of chromatin loop domains, but proceeded without internucleosomal DNA cleavage. In contrast to apoptosis associated with internucleosomal DNA fragmentation, the apoptotic pathway associated with excision of chromatin loop domains was largely caspase independent. We identify here MAPK family member, p38/JNK, mitochondria, and topoisomerase II as the components of this caspase-independent apoptotic pathway. While caspase-independent excision of chromatin loop domains was a predominant mechanism of DNA disintegration in staurosporine-treated neuroblastoma, both caspase-dependent internucleosomal DNA fragmentation and caspase-independent excision of chromatin loop domains accompanied staurosporine-induced apoptosis of promyelocytic leukemia cells. Our results suggest that caspase-independent excision of chromatin loop domains represents a separate cell death pathway, which operates either in parallel or independently from caspase-dependent internucleosomal DNA fragmentation.