Abstract 7245: Mechanistic dissection of ABI1 as DNA-binding transcriptional regulator in cancer cells

Background: Prostate cancer remains a challenging issue affecting men worldwide. In contrast to localized disease, metastatic prostate cancer tumor is incurable. Progression to metastatic disease is characterized by tumor heterogeneity and plasticity regulated by epigenetic changes of tumor cells. While tumor heterogeneity is often associated with gene mutations, tumor plasticity is ascribed to transcriptional regulation and chromatin activity. The latter is modulated by treatment of prostate cancer leading to treatment resistance. Here, we identified a novel mechanism of epigenetic regulation in prostate cancer by an actin regulatory and signaling protein, Abelson Interactor 1 (ABI1). Methods: To demonstrate the ABI1-DNA binding, we employed in vitro binding assays, NMR spectroscopy, and ChIP-seq. from cells expressing alternatively spliced isoforms of ABI1 that contain or lack the DNA binding HHR domain. To determine the ABI1 interactions with several transcriptional factors, we used immunoprecipitation and/or proximity ligation assay (PLA). We used ATAC sequencing to determine the chromatin accessibility in the presence or absence of ABI1 gene. RNA-seq established the activity of transcriptional factors and signaling pathways regulated by ABI1. Results: Using NMR spectroscopy, we established that ABI1 demonstrated DNA binding dependent on the presence of alternatively spliced Exon 4. Exon 4 is located within the homeobox homology region (HHR). The DNA binding activity was confirmed using in vitro binding assays; the assay results confirmed the role of ABI1 HHR. Moreover, cells lacking ABI1 or expressing ABI1 lacking Exon 4 show low accessibility and activity of chromatin. In contrast, cells expressing ABI1 containing Exon 4 show active chromatin. RNA-seq analysis demonstrated that ABI1 regulated transcription in an Exon 4-dependent manner. Conclusions: We uncovered a role of ABI1 in chromatin stability, that is dependent on the presence of the DNA binding region. Notably, ABI1's contribution to transcriptional activity occurs via a transient yet precisely defined interaction between its intrinsically disordered region and DNA. This regulatory control plays a pivotal role in managing tumor plasticity by establishing connections between the actin cytoskeleton, cellular signaling, and transcriptional regulation. Based on these findings, we propose that ABI1 functions as an epigenetic regulator, maintaining transcriptional homeostasis in prostate cancer. Citation Format: Leszek Kotula. ABI1 regulates prostate tumor plasticity through chromatin remodeling and transcriptional regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1701.

Introduction and Objective: The serine/threonine kinases of the protein kinase D family (PKCmu/PKD1, PKD2, PKCnu/PKD3), a subfamily of the CAMK superfamily, have been implicated in regulation of multiple biological processes including proliferation, survival, apoptosis, angiogenesis and motility. The precise mechanisms by which the three PKDs modulate these processes are incompletely understood and require a better knowledge of their signaling context. Methodology and Results: Using the ProtoArray Human Protein Microarray v4.0 (Invitrogen), we identified Abelson interactor 1 (ABI1) as a novel PKD2 substrate. ABI1 was first identified as the downstream target of Abl tyrosine kinase and is an adaptor protein involved in actin reorganization and lamellipodia formation along with WAVE2. In the present study we illustrate that PKD2 interacts and phosphorylates ABI1 in vitro and in vivo at two potential serine residues. The sites were validated using an antibody that specifically identifies the consensus PKD substrate sequence. Furthermore, PKD2-induced phosphorylation of ABI1 destabilized the interaction between ABI1 and WAVE2 and resulted in relocation of both the proteins from plasma membrane to perinuclear region. Consequences of this destabilization and its potential impact on WAVE2 driven actin polymerization and the associated processes are in the process of investigation. Preliminary results also suggest reduced migration velocity and directedness of a tumor cell. Conclusion: In conclusion, our data demonstrate that ABI1 is a novel substrate of PKD2 that might negatively regulate tumor cell migration via terminating a constitutive interaction between ABI1 and WAVE2. *Impact of ABI1-WAVE2 subcomplex destabilization on WAVE2 induced actin polymerization and lamellipodia extension will be presented at the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-513. doi:1538-7445.AM2012-LB-513

Prostate cancer is the most common non-skin cancer among men worldwide and the second leading cause of cancer-related deaths. Prostate tumor development is driven by androgen receptor (AR) signaling, and while early-stage prostate cancer responds well to anti-androgen therapies, resistance frequently emerges. This resistance often involves aberrant reactivation of AR signaling, underscoring the critical need for a deeper understanding of AR regulation to inform new therapeutic strategies.AR activation is mediated by the intrinsically disordered N-terminal domain (NTD), which contains several proline-rich (PXXP) motifs that serve as consensus sequences for SH3 domain binding. Our previous work identified Abelson-interactor 1 (ABI1) as a novel AR-binding protein via ABI1's SH3 domain. ABI1, a key regulator of the actin cytoskeleton as part of the WAVE complex, exhibits dysregulated expression during prostate cancer progression. We hypothesize that ABI1 regulates AR, and its splice variant AR-V7, through multivalent interactions between ABI1's SH3 domain and PXXP motifs in the AR NTD. To investigate this, we used purified AR PXXP peptides and ABI1-SH3 probes in far-western blotting assays, demonstrating that ABI1-SH3 preferentially binds to two of the seven canonical PXXP motifs within AR-NTD. In vivo studies with AR NTD-truncation mutants further confirmed that ABI1 regulates AR activity through these interactions. These findings position ABI1 as a regulator of AR activity, potentially linking AR dysregulation with cytoskeletal reorganization and cancer cell metastasis. This work enhances our understanding of ABI1's role in AR-driven tumorigenesis and provides a foundation for future studies aimed at targeting ABI1-AR interactions to develop novel therapies for advanced prostate cancer. Citation Format: Kevin Mengen Lin, David M. Solano, Baylee Porter-Hansen, Xiang Li, Leszek Kotula. Defining the mechanism of androgen receptor activation by ABI1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1417.

CUTL1, also known as CDP (CCAAT Displacement Protein), Cut, or Cux-1, is a homeodomain transcription factor known to play an essential role in development and cell cycle progression. Previously, we identified CUTL1 as modulator of cell motility and invasiveness. Here we report that protein kinase A (PKA), known to inhibit tumor progression in various tumor types, directly phosphorylates CUTL1 at serine 1215 in NIH3T3 fibroblasts. The PKA-induced phosphorylation results in decreased DNA binding affinity of CUTL1 and diminished CUTL1-mediated cell cycle progression and cell motility. Furthermore, the expression of several CUTL1 target genes involved in proliferation and migration, such as DNA polymerase A and DKK2, was modulated by PKA-induced phosphorylation. These data identify CUTL1 as a novel target of PKA through which this protein kinase can modulate tumor cell motility and tumor progression.

A Double Interaction Screen identifies positive and negative ftz gene regulators and Ftz-interacting proteins

EKLF/KLF-1 is an erythroid-restricted transcription factor essential for expression of the adult beta-globin gene. EKLF/KLF-1 is a 358-amino acid nuclear protein with an amino-terminal proline-rich domain and a carboxyl-terminal DNA binding domain. The nuclear localization signal (NLS) of EKLF/KLF-1 has not been empirically determined. We generated a series of epitope-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to the 83-amino acid (amino acids 276-358) DNA binding domain that consists of three Kruppel zinc fingers. All three zinc fingers are necessary for efficient nuclear localization; deletion of any individual finger results in cytoplasmic accumulation. Fusion of the three zinc fingers to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. EKLF/KLF-1 containing histidine to alanine mutations that disrupt the structure of all three fingers retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. We demonstrate that basic residues within the fingers are the critical determinants for nuclear localization; mutations of these basic residues to alanine resulted in cytoplasmic mislocalization. The basic residues of all mammalian Kruppel zinc fingers are highly conserved; therefore we propose that these basic residues are a common NLS shared by all Kruppel family members.

Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.

The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1beta. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBPbeta carboxyl-terminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity.

In Methanobacterium thermoautotrophicum, the fmdECB operon encoding the molybdenum formyl-methanofuran dehydrogenase is directly preceded by an open reading frame tfx predicted to encode a DNA binding protein. The 16.1 kDa protein has an N-terminal basic domain with a helix-turn-helix motif for DNA binding and a C-terminal acidic domain possibly for transcriptional activation. We report here on the DNA binding properties of the Tfx protein heterologously overproduced in Escherichia coli. Tfx was found to bind specifically to a DNA sequence downstream of the promoter of the fmdECB operon, as shown by electrophoretic mobility shift assays and DNase I footprint analysis. Northern blot hybridizations revealed that transcription of tfx is repressed during the growth of M. thermoautotrophicum in the presence of tung-state. Based on its structure and properties, the DNA binding protein Tfx is proposed to be a transcriptional regulator composed of a basic DNA binding domain and an acidic activation domain.

The transcriptional activity of androgen receptor (AR) is modulated by coregulators that have a significant influence on a number of functional properties of AR, including the ligand specificity as well as the DNA binding capacity. Because androgens and AR have pivotal roles in the development and

Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.

hSSB1 (human single strand DNA-binding protein 1) has been shown to participate in homologous recombination (HR)-dependent repair of DNA double strand breaks (DSBs) and ataxia telangiectasia-mutated (ATM)-mediated checkpoint pathways. Here we present evidence that hSSB2, a homolog of hSSB1, plays a role similar to hSSB1 in DNA damage-response pathways. This was evidenced by findings that hSSB2-depleted cells resemble hSSB1-depleted cells in hypersensitivity to DNA-damaging reagents, reduced efficiency in HR-dependent repair of DSBs, and defective ATM-dependent phosphorylation. Notably, hSSB1 and hSSB2 form separate complexes with two identical proteins, INTS3 and hSSBIP1 (C9ORF80). Cells depleted of INTS3 and hSSBIP1 also exhibited hypersensitivity to DNA damage reagents, chromosomal instability, and reduced ATM-dependent phosphorylation. hSSBIP1 was rapidly recruited to laser-induced DSBs, a feature also similar to that reported for hSSB1. Depletion of INTS3 decreased the stability of hSSB1 and hSSBIP1, suggesting that INTS3 may provide a scaffold to allow proper assembly of the hSSB complexes. Thus, our data demonstrate that hSSB1 and hSSB2 form two separate complexes with similar structures, and both are required for efficient HR-dependent repair of DSBs and ATM-dependent signaling pathways.

Nuclear factor (NF)-kappaB transcription factors are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. Transcription of the human immunodeficiency virus type 1 (HIV-1) genome depends on the intracellular environment where the integrate viral DNA is regulated by a complex interplay among viral regulatory proteins, such as Tat, and host cellular transcription factors, such as NF-kappaB, interacting with the viral long terminal repeat region. CBP (CREB-binding protein) and p300, containing an intrinsic histone acetyltransferase (HAT) activity, have emerged as coactivators for various DNA-binding transcription factors. Here, we show that the p50 subunit as well as the p50/p65 of NF-kappaB, and not other factors such as SP1, TFIIB, polymerase II, TFIIA, or p65, can be acetylated by CBP/p300 HAT domain. Acetylation of p50 was completely dependent on the presence of both HAT domain and Tat proteins, implying that Tat influences the transcription machinery by aiding CBP/p300 to acquire new partners and increase its functional repertoire. Three lysines, Lys-431, Lys-440, and Lys-441 in p50 were all acetylated in vitro, and a sequence similarity among p50, p53, Tat, and activin receptor type I on these particular lysines was observed. All proteins have been shown to be acetylated by the CBP/p300 HAT domain. Acetylated p50 increases its DNA binding properties, as evident by streptavidin/biotin pull-down assays when using labeled NF-kappaB oligonucleotides. Increased DNA binding on HIV-1 long terminal repeat coincided with increases in the rate of transcription. Therefore, we propose that acetylation of the DNA binding domain of NF-kappaB aids in nuclear translocation and enhanced transcription and also suggest that the substrate specificity of CBP/p300 can be altered by small peptide molecules, such as HIV-encoded Tat.

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.