TRIM66 reads unmodified H3R2K4 and H3K56ac to respond to DNA damage in embryonic stem cells

Recognition of specific chromatin modifications by distinct structural domains within “reader” proteins plays a critical role in the maintenance of genomic stability. However, the specific mechanisms involved in this process remain unclear. Here we report that the PHD-Bromo tandem domain of tripartite motif-containing 66 (TRIM66) recognizes the unmodified H3R2-H3K4 and acetylated H3K56. The aberrant deletion of Trim66 results in severe DNA damage and genomic instability in embryonic stem cells (ESCs). Moreover, we find that the recognition of histone modification by TRIM66 is critical for DNA damage repair (DDR) in ESCs. TRIM66 recruits Sirt6 to deacetylate H3K56ac, negatively regulating the level of H3K56ac and facilitating the initiation of DDR. Importantly, Trim66-deficient blastocysts also exhibit higher levels of H3K56ac and DNA damage. Collectively, the present findings indicate the vital role of TRIM66 in DDR in ESCs, establishing the relationship between histone readers and maintenance of genomic stability.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Nikon A1R (Nikon) was used to acquire confocal images.

Data analysis
Prism version 6 software (GraphPad) was used to generate graphs and perform statistical analyses; ImageQuant LAS400 was used for WB analyses; NIS-Elements C (Nikon) sofware was used for immunofluorescence confocal microscopy analysis; ImageJ was used for quantifying the fluorescence intensity; MicroCal PEAQ-ITC analysis software was used for ITC results analyses.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The atomic coordinates and structure factors for the TRIM66 in free state and in the complex have been deposited to the Protein Data Bank (PDB) under the accession code PDB 6IET and 6IEU.All additional data that support the findings of this study are available from the corresponding author upon reasonable request. Uncropped gel images and other source data underlying Figs 3a-c, e and g, 4c and e-h, 5a-c, e and g-j, 6a-c and e-j, 7b, e and h, Supplementary Figs 1e Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
There was no sample size calculation.
Data exclusions No data was excluded.

Replication
All attempts at replication of the results in this study were successful.
Randomization Samples for assay were randomly allocated.

Blinding
Blinding was used for all data acquisition and analysis.

Behavioural & social sciences study design
All studies must disclose on these points even when the disclosure is negative.

Study description
Briefly describe the study type including whether data are quantitative, qualitative, or mixed-methods (e.g. qualitative cross-sectional, quantitative experimental, mixed-methods case study).

Data collection
Provide details about the data collection procedure, including the instruments or devices used to record the data (e.g. pen and paper, computer, eye tracker, video or audio equipment) whether anyone was present besides the participant(s) and the researcher, and whether the researcher was blind to experimental condition and/or the study hypothesis during data collection.

Timing
Indicate the start and stop dates of data collection. If there is a gap between collection periods, state the dates for each sample cohort.

Data exclusions
If no data were excluded from the analyses, state so OR if data were excluded, provide the exact number of exclusions and the rationale behind them, indicating whether exclusion criteria were pre-established.

Non-participation
State how many participants dropped out/declined participation and the reason(s) given OR provide response rate OR state that no participants dropped out/declined participation.

Randomization
If participants were not allocated into experimental groups, state so OR describe how participants were allocated to groups, and if allocation was not random, describe how covariates were controlled.

Ecological, evolutionary & environmental sciences study design
All studies must disclose on these points even when the disclosure is negative.

Describe any disturbance caused by the study and how it was minimized.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Authentication Describe the authentication procedures for each cell line used OR declare that none of the cell lines used were authenticated.

Mycoplasma contamination
All cell lines tested are free for mycoplasma contamination.

Commonly misidentified lines (See ICLAC register)
Not present in the study Palaeontology Specimen provenance Provide provenance information for specimens and describe permits that were obtained for the work (including the name of the issuing authority, the date of issue, and any identifying information).

Specimen deposition
Indicate where the specimens have been deposited to permit free access by other researchers.

Dating methods
If new dates are provided, describe how they were obtained (e.g. collection, storage, sample pretreatment and measurement), where they were obtained (i.e. lab name), the calibration program and the protocol for quality assurance OR state that no new dates are provided.
Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
We used mice of mixed strain that were two months old at the beginning of each experiment, males and females were used in all experiments with indistinguishable results.

Wild animals
No wild animals were used

Field-collected samples
No field collected sample were used

Ethics oversight
All animal experiments were approved by the Institutional Animal Care Committee of Tongji University under the Guide for the Care and Use of Laboratory Animals (NIH Guide). All mice were maintained in a pathogen-free environment throughout the experiments, and all efforts were made to minimize the number of animals used and their suffering.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants

Data quality
Describe the methods used to ensure data quality in full detail, including how many peaks are at FDR 5% and above 5-fold enrichment.

Software
Describe the software used to collect and analyze the ChIP-seq data. For custom code that has been deposited into a community repository, provide accession details.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
Describe the sample preparation, detailing the biological source of the cells and any tissue processing steps used.

Instrument
Identify the instrument used for data collection, specifying make and model number.

Software
Describe the software used to collect and analyze the flow cytometry data. For custom code that has been deposited into a community repository, provide accession details.
Cell population abundance Describe the abundance of the relevant cell populations within post-sort fractions, providing details on the purity of the samples and how it was determined.

Gating strategy
Describe the gating strategy used for all relevant experiments, specifying the preliminary FSC/SSC gates of the starting cell population, indicating where boundaries between "positive" and "negative" staining cell populations are defined.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary  Design specifications Specify the number of blocks, trials or experimental units per session and/or subject, and specify the length of each trial or block (if trials are blocked) and interval between trials.