Periodic Reporting for period 2 - NucEM (Accessing Nucleosomal DNA)
Período documentado: 2022-09-01 hasta 2024-02-29
• What is the problem/issue being addressed?
Differential gene expression programs are controlled by intricate networks of sequence-specific transcription factors (TFs) that operate in the context of chromatin. Chromatin is itself an essential component of this process and is part of a system that selectively restricts DNA access for TFs. As a consequence, most TFs only bind to a small subset of their motif occurrences, in a way we currently do not fully understand. Chromatin is comprised of multiple nucleosomes, each containing two copies of histones H2A, H2B, H3 and H4 together with ca. 146 base pairs duplex DNA. When wrapped around the nucleosome, the solvent accessibility of the packaged DNA is severely restricted, and this barrier needs to be overcome for transcription to occur. How proteins contact the largely occluded nucleosomal DNA in a sequence-specific manner and whether the nucleosome assists in motif selection is unclear, and will be addressed in the context of this ERC grant.
• Why is it important for society?
We aim to provide the basic rules governing TF engagement to DNA on nucleosomes. Inadvertent gene activation in cancer remains a problem of large biomedical relevance and most of the TFs covered in this proposal function as oncogenes and are considered undruggable. The envisaged structure/function study is aimed to reveal the molecular determinants governing chromatin binding for TFs, which has thus far not been attempted. Obtaining structures across structurally diverse nucleosome binders will clarify to what extent TFs leverage nucleosomes as part of their sequence read-out, leading to a better understanding of TF action in health and disease. While it is challenging to find small molecule binders/inhibitors for TFs, complex formation between TFs and nucleosomes may also provide additional, suitable, pockets for small molecule drug discovery. A better molecular understanding of these TFs and their mode of action in the context of chromatin, their natural habitat, will hence also ultimately lead to better drugs targeting bHLH TFs.
• What are the overall objectives?
Tissue-specific variation in the expression of ~1600 human transcription factors provides the basis for multicellularity. Yet DNA sequence alone is a weak predictor of TF occupancy. Instead, one of the most predictive features of TF DNA binding is reduced nucleosomal presence, illustrating how chromatin limits TF engagement. The key questions in the field are how nucleosomes impact TF DNA accessibility, how single TFs engage chromatinised binding sites, and how multiple TFs cooperate to read out nucleosome-occluded motifs.
Differential gene expression programs are controlled by intricate networks of sequence-specific transcription factors (TFs) that operate in the context of chromatin. Chromatin is itself an essential component of this process and is part of a system that selectively restricts DNA access for TFs. As a consequence, most TFs only bind to a small subset of their motif occurrences, in a way we currently do not fully understand. Chromatin is comprised of multiple nucleosomes, each containing two copies of histones H2A, H2B, H3 and H4 together with ca. 146 base pairs duplex DNA. When wrapped around the nucleosome, the solvent accessibility of the packaged DNA is severely restricted, and this barrier needs to be overcome for transcription to occur. How proteins contact the largely occluded nucleosomal DNA in a sequence-specific manner and whether the nucleosome assists in motif selection is unclear, and will be addressed in the context of this ERC grant.
• Why is it important for society?
We aim to provide the basic rules governing TF engagement to DNA on nucleosomes. Inadvertent gene activation in cancer remains a problem of large biomedical relevance and most of the TFs covered in this proposal function as oncogenes and are considered undruggable. The envisaged structure/function study is aimed to reveal the molecular determinants governing chromatin binding for TFs, which has thus far not been attempted. Obtaining structures across structurally diverse nucleosome binders will clarify to what extent TFs leverage nucleosomes as part of their sequence read-out, leading to a better understanding of TF action in health and disease. While it is challenging to find small molecule binders/inhibitors for TFs, complex formation between TFs and nucleosomes may also provide additional, suitable, pockets for small molecule drug discovery. A better molecular understanding of these TFs and their mode of action in the context of chromatin, their natural habitat, will hence also ultimately lead to better drugs targeting bHLH TFs.
• What are the overall objectives?
Tissue-specific variation in the expression of ~1600 human transcription factors provides the basis for multicellularity. Yet DNA sequence alone is a weak predictor of TF occupancy. Instead, one of the most predictive features of TF DNA binding is reduced nucleosomal presence, illustrating how chromatin limits TF engagement. The key questions in the field are how nucleosomes impact TF DNA accessibility, how single TFs engage chromatinised binding sites, and how multiple TFs cooperate to read out nucleosome-occluded motifs.
We have thus far shown how OCT4 and SOX2, two pioneer TFs essential for stem cell pluripotency and capable of reprogramming differentiated cells, access joint motifs in the context of a nucleosome. We developed an assay to determine the accessibility profile for OCT4/SOX2 at base-pair resolution throughout an entire nucleosome. This revealed preferred binding at the entry/exit sites of nucleosomal DNA enabling structural analysis in order to dissect the molecular mechanism. The resulting cryo-EM structures of OCT4-SOX2 bound nucleosome of two representative motif locations are, to our knowledge, the first structures of a TF engaging a nucleosome, revealing novel principles that govern TF access throughout chromatin. The structural and functional dissection reveals that OCT4 and SOX2 together remodel the nucleosome and distort the DNA trajectory, without changing the histone octamer core.
We have extended this analysis now to a second family of transcription factors, the basic helix-loop-helix (bHLH) TF family whose members are able to bind a generic CANNTG DNA motif (known as E-boxes) occurring ~15 mio times in the human genome. Only a fraction (~1%) of E-boxes are actually occupied at given time, and the principles governing binding were unknown. Our work focused on two phylogenetically and structurally diverse members: the bHLH leucine-zipper (bHLH-LZ) TF, MYC-MAX, a key regulator of cell proliferation and oncogene, and the bHLH PAS-domain containing (bHLH-PAS) TF, CLOCK-BMAL1, an important transcriptional circuit driver of the cellular circadian clock. While CLOCK-BMAL1 has been linked to opening and occupying chromatinised sites as a pioneer factor, MYC-MAX is assumed to be dependent on other proteins to access chromatin. We find that MYC-MAX and CLOCK-BMAL1 preferentially bind E-boxes near the ends of the nucleosomal DNA, yet show different accessibility profiles depending on the dimerization domains present. Structural studies including endogenous nucleosome positioning sequences such as Lin28 demonstrate that MYC-MAX, MAX-MAX and CLOCK-BMAL1 release DNA from the histones to gain access to nucleosomes, clarifying a previously controversial readout mechanism. We observe unexpected and extensive histone interactions between the bHLH-dimerization domains, particularly for CLOCK-BMAL1. This work introduces TF/histone interactions as important and ubiquitous specifier of bHLH binding. These findings challenge the notion of unique TFs/nucleosome contact interfaces, and instead establish that different motif registers fine-tune bHLH affinity for nucleosomes through different interfaces, as well as determine the extent of competition with other proteins for nucleosome access.
We have extended this analysis now to a second family of transcription factors, the basic helix-loop-helix (bHLH) TF family whose members are able to bind a generic CANNTG DNA motif (known as E-boxes) occurring ~15 mio times in the human genome. Only a fraction (~1%) of E-boxes are actually occupied at given time, and the principles governing binding were unknown. Our work focused on two phylogenetically and structurally diverse members: the bHLH leucine-zipper (bHLH-LZ) TF, MYC-MAX, a key regulator of cell proliferation and oncogene, and the bHLH PAS-domain containing (bHLH-PAS) TF, CLOCK-BMAL1, an important transcriptional circuit driver of the cellular circadian clock. While CLOCK-BMAL1 has been linked to opening and occupying chromatinised sites as a pioneer factor, MYC-MAX is assumed to be dependent on other proteins to access chromatin. We find that MYC-MAX and CLOCK-BMAL1 preferentially bind E-boxes near the ends of the nucleosomal DNA, yet show different accessibility profiles depending on the dimerization domains present. Structural studies including endogenous nucleosome positioning sequences such as Lin28 demonstrate that MYC-MAX, MAX-MAX and CLOCK-BMAL1 release DNA from the histones to gain access to nucleosomes, clarifying a previously controversial readout mechanism. We observe unexpected and extensive histone interactions between the bHLH-dimerization domains, particularly for CLOCK-BMAL1. This work introduces TF/histone interactions as important and ubiquitous specifier of bHLH binding. These findings challenge the notion of unique TFs/nucleosome contact interfaces, and instead establish that different motif registers fine-tune bHLH affinity for nucleosomes through different interfaces, as well as determine the extent of competition with other proteins for nucleosome access.
Our work shows that the same motif in a different rotational/translation setting on a nucleosome, gives rise to different structures/binding modes. We also find that the DNA binding motif employed by OCT4-SOX2 on a nucleosome differs from that observed on naked DNA, a binding mode that accounts for their genomic binding. This long sought-after structural snapshot of TF-induced DNA release from the nucleosome provides a first explanation how pioneer factor function, and suggests a conceptual framework for how TFs read out nucleosomal DNA, which we will explore and confirm for other TF families in the future.
Key questions in the field are how nucleosomes impact TF DNA accessibility, how single TFs engage chromatinised binding sites, and how multiple TFs cooperate to read out nucleosome-occluded motifs. We now provide first insights into all of these aspects for individual TFs. A better molecular understanding of these TFs and their mode of action in the context of chromatin, their natural chromatin habitat, will ultimately lead to better drugs targeting TFs in cancer therapy and beyond.
Key questions in the field are how nucleosomes impact TF DNA accessibility, how single TFs engage chromatinised binding sites, and how multiple TFs cooperate to read out nucleosome-occluded motifs. We now provide first insights into all of these aspects for individual TFs. A better molecular understanding of these TFs and their mode of action in the context of chromatin, their natural chromatin habitat, will ultimately lead to better drugs targeting TFs in cancer therapy and beyond.