Periodic Reporting for period 4 - HybReader (Mechanisms of epigenetic gene regulation by R-loops)
Reporting period: 2022-12-01 to 2023-11-30
R-loops, three-stranded DNA-RNA hybrids, are formed by invasion of an RNA strand into duplex DNA and can be assembled either co-transcriptionally (cis R-loops) or by local unwinding of DNA and subsequent hybridization of homologous RNA fragments (trans R-loops). R-loops have long been regarded as unwanted structures, potentially leading to transcription or replication fork collisions as well as leading to a single DNA strand, which is energetically less stable than double stranded DNA and therefore prone to damaging agents. Recent advances have, however, challenged this view and shown a subclass of R-loops to be involved in epigenetic regulation of a number of genes, leading to R-loops as being often described as a double-edged sword: A threat to genome integrity (unscheduled R-loops) and epigenetic regulators of gene expression (regulatory R-loops).
Regulatory RNAs play key roles in development and disease and yet we are still at the beginning of understanding their biology and mode of action. Among regulatory RNAs, R-loops, prevalent DNA:RNA hybrids, which lie at the interface of different nuclear processes and neurodegenerative disease, have come into the limelight as novel gene regulators. Accumulating evidence indicates that lncRNAs can act as guide RNAs to target gene regulators to genes via R-loops. This is a breakthrough in understanding how these DNA:RNA hybrids act as signals and sets the stage to address fundamental questions regarding the epigenetic mechanisms of R-loop function and their biology:
• What is the function of regulatory R-loops?
• Do they form via cis- or trans acting RNAs?
• How are the potentially detrimental effects of regulatory R-loops constrained?
• How are regulatory R-loops regulated?
• What molecular determinants distinguish regulatory R-loops from unscheduled R-loops?
• How is the information of regulatory R-loops coded and decoded?
The objective of this project is to provide answers to these questions by making use of an epigenetic R-loop reader protein, Gadd45a, which we have identified previously.
The growth arrest and DNA damage inducible 45 (GADD45) family of proteins has three members: GADD45a, GADD45b, GADD45g. These small proteins (~18 kDa) share high sequence similarity and function in regulating stress response, DNA damage repair and cell cycle control. GADD45 proteins lack enzymatic activity and carry out their functions by recruiting enzymatic effectors. Intriguingly, recent work from our lab revealed GADD45a to function as an R-loop reader. The study showed that the TARID lncRNA (TCF21 Antisense RNA Inducing Demethylation) binds to the TCF21 gene promoter forming an R-loop. GADD45a binds this R-loop and in turn recruits TET1 and TDG to bring about active DNA demethylation and activate the TCF21 gene transcription. However, mechanistic insight into genome-wide GADD45a-mediated R-loop decoding is still missing.
Key tools have been obtained during the action, including a genome-edited Gadd45a gene harbouring an HA-epitope for facilitated Gadd45a detection as well as several Gadd45a,b,g triple mutant ESC lines.
A vast amount of data was obtained during the action, including genome-wide profilings to characterize the nuclear role of Gadd45. Only a minor fraction of the data has been published as yet. Comprehensive papers reporting these results will be released in the coming years. We anticipate that by the time of publication, we will have an understanding of the role of Gadd45 decoding regulatory R-loop hybrids in undifferentiated and differentiating mouse embryonic stem cells.
Regulatory RNAs play key roles in development and disease and yet we are still at the beginning of understanding their biology and mode of action. Among regulatory RNAs, R-loops, prevalent DNA:RNA hybrids, which lie at the interface of different nuclear processes and neurodegenerative disease, have come into the limelight as novel gene regulators. Accumulating evidence indicates that lncRNAs can act as guide RNAs to target gene regulators to genes via R-loops. This is a breakthrough in understanding how these DNA:RNA hybrids act as signals and sets the stage to address fundamental questions regarding the epigenetic mechanisms of R-loop function and their biology:
• What is the function of regulatory R-loops?
• Do they form via cis- or trans acting RNAs?
• How are the potentially detrimental effects of regulatory R-loops constrained?
• How are regulatory R-loops regulated?
• What molecular determinants distinguish regulatory R-loops from unscheduled R-loops?
• How is the information of regulatory R-loops coded and decoded?
The objective of this project is to provide answers to these questions by making use of an epigenetic R-loop reader protein, Gadd45a, which we have identified previously.
The growth arrest and DNA damage inducible 45 (GADD45) family of proteins has three members: GADD45a, GADD45b, GADD45g. These small proteins (~18 kDa) share high sequence similarity and function in regulating stress response, DNA damage repair and cell cycle control. GADD45 proteins lack enzymatic activity and carry out their functions by recruiting enzymatic effectors. Intriguingly, recent work from our lab revealed GADD45a to function as an R-loop reader. The study showed that the TARID lncRNA (TCF21 Antisense RNA Inducing Demethylation) binds to the TCF21 gene promoter forming an R-loop. GADD45a binds this R-loop and in turn recruits TET1 and TDG to bring about active DNA demethylation and activate the TCF21 gene transcription. However, mechanistic insight into genome-wide GADD45a-mediated R-loop decoding is still missing.
Key tools have been obtained during the action, including a genome-edited Gadd45a gene harbouring an HA-epitope for facilitated Gadd45a detection as well as several Gadd45a,b,g triple mutant ESC lines.
A vast amount of data was obtained during the action, including genome-wide profilings to characterize the nuclear role of Gadd45. Only a minor fraction of the data has been published as yet. Comprehensive papers reporting these results will be released in the coming years. We anticipate that by the time of publication, we will have an understanding of the role of Gadd45 decoding regulatory R-loop hybrids in undifferentiated and differentiating mouse embryonic stem cells.
The following analyses have been conducted in the project so far:
• 4sU-seq on “Gadd45 TKO 1.0” to investigate differences in levels of nascent transcripts of heat shock genes upon thermal stress.
• Mapping the GADD45a and GADD45b binding sites in mESCs under the thermal stress conditions.
• Cell fractionation of Gadd45a protein to identify nucleo-cytoplasmic ratio in ESCs. The protein is mostly cytoplasmic.
• Generating a genome-edited Gadd45a gene harbouring an HA-epitope for facilitated Gadd45a detection. IF microscopy analysis of Gadd45a protein done.
• Tested several genome-wide R-loop mapping protocols and found spKas and S9.6 Cut & Run DRIP-seq protocols to work successfully. Thereby mapped R-loops genome-wide in mESCs.
• Generated and validated a “2nd“ and “3rd“-generation (TKO 3.0)” Gadd45a,b,g triple mutant mESCs cell lines.
• Generated and validated a “4th“-generation (TKO 4.0)” Gadd45a,b,g triple mutant mESCs cell line in a GFP-expressing Rosa-eGFP mESC line.
• RNA exosome ChIP-seq analysis in Wt and “Gadd45 TKO 2.0”.
• CoIP exepriments between Gadd45a and i) RNA exosome components and ii) Tip60/p400.
• Analyzed bioinformatically lncRNAs in relation to Gadd45a-bound sites. Correlated lncRNA sites to the presence of R-loops and Tet1 bindings sites.
• Characterization of Gadd45 TKO 3.0 is in progress (RNA-seq, Pol II mapping, Tip60/p400 ChIP-seq, chromatin mark ChIP-seq, Tet1 ChIP-seq, NELF-seq, ATAC-seq, ssRNA-seq, S9.6 Cut & Run DRIP-seq for R-loop mapping).
• Bioinformatic data analyses and data integration of these Omics-datasets.
• Genome-wide analysis of Gadd45a binding sites obtained from incubating naked gDNA with recombinant Gadd45a protein.
• Performing EMSA using purified GADD45a and GADD45b on selected DNA:RNA hybrids.
• Established a mESC in vitro differentiation protocol for cardiac differentiation. Established FACS and qPCR analysis for the same.
• Conducted mESC cardiac in vitro differentiation with wt and Gadd45 TKO 3.0 mESCs, characterized the TKO-associated differentiation defects.
Parts of these Results have been reported at meetings. Only a minor fraction has been published as yet. Comprehensive papers reporting these results will be published in the coming years. We anticipate that by the time of publication, we will have an understanding of the role of Gadd45a decoding regulatory R-loop hybrids in undifferentiated and differentiating mouse embryonic stem cells.
• 4sU-seq on “Gadd45 TKO 1.0” to investigate differences in levels of nascent transcripts of heat shock genes upon thermal stress.
• Mapping the GADD45a and GADD45b binding sites in mESCs under the thermal stress conditions.
• Cell fractionation of Gadd45a protein to identify nucleo-cytoplasmic ratio in ESCs. The protein is mostly cytoplasmic.
• Generating a genome-edited Gadd45a gene harbouring an HA-epitope for facilitated Gadd45a detection. IF microscopy analysis of Gadd45a protein done.
• Tested several genome-wide R-loop mapping protocols and found spKas and S9.6 Cut & Run DRIP-seq protocols to work successfully. Thereby mapped R-loops genome-wide in mESCs.
• Generated and validated a “2nd“ and “3rd“-generation (TKO 3.0)” Gadd45a,b,g triple mutant mESCs cell lines.
• Generated and validated a “4th“-generation (TKO 4.0)” Gadd45a,b,g triple mutant mESCs cell line in a GFP-expressing Rosa-eGFP mESC line.
• RNA exosome ChIP-seq analysis in Wt and “Gadd45 TKO 2.0”.
• CoIP exepriments between Gadd45a and i) RNA exosome components and ii) Tip60/p400.
• Analyzed bioinformatically lncRNAs in relation to Gadd45a-bound sites. Correlated lncRNA sites to the presence of R-loops and Tet1 bindings sites.
• Characterization of Gadd45 TKO 3.0 is in progress (RNA-seq, Pol II mapping, Tip60/p400 ChIP-seq, chromatin mark ChIP-seq, Tet1 ChIP-seq, NELF-seq, ATAC-seq, ssRNA-seq, S9.6 Cut & Run DRIP-seq for R-loop mapping).
• Bioinformatic data analyses and data integration of these Omics-datasets.
• Genome-wide analysis of Gadd45a binding sites obtained from incubating naked gDNA with recombinant Gadd45a protein.
• Performing EMSA using purified GADD45a and GADD45b on selected DNA:RNA hybrids.
• Established a mESC in vitro differentiation protocol for cardiac differentiation. Established FACS and qPCR analysis for the same.
• Conducted mESC cardiac in vitro differentiation with wt and Gadd45 TKO 3.0 mESCs, characterized the TKO-associated differentiation defects.
Parts of these Results have been reported at meetings. Only a minor fraction has been published as yet. Comprehensive papers reporting these results will be published in the coming years. We anticipate that by the time of publication, we will have an understanding of the role of Gadd45a decoding regulatory R-loop hybrids in undifferentiated and differentiating mouse embryonic stem cells.
The analyses expanded to GADD45 proteins and the RNA exosome, revealing potential protein partners, including RNA exosome components. Efforts extended to generating Gadd45a,b,g triple mutant ESCs. Initial cell lines had issues like residual Gadd45a expression and chromosomal aberrations, prompting repeated generation and thorough characterization of a 3rd generation Gadd45a,b,g triple mutant ESC line (Gadd45 TKO 3.0 mESCs). Studies uncovered cardiomyocyte differentiation defects in Gadd45 TKO 3.0 mESCs, shedding light on Gadd45's role in heart development. Ongoing investigations aim to elucidate Gadd45a's involvement in heart development and R-loop binding in gene promoters. We also established several protocols for genome-wide R-loop mapping in ESCs and document the location of these structures strand specifically. Taken together, this research, presented at various meetings, contributes to understanding the role of Gadd45 as R-loop reader, its role in embryonic cell differentiation, and in organismic aging. Comprehensive papers reporting these roles will be published in the coming years.