Histone ubiquitylation interactome mapping

The interactome of H2BK120ub

Previously in our laboratory, using chromatin affinity purification coupled with quantitative mass spectrometry, the interactome of H2BK120ub was presented [83]. In that study, ubiq-uitylation recruited to chromatin the RNA polymerase, proteins that control transcription elongation, the small nuclear RNA-processing integrator complex and the switch/sucrose nonfermentable chromatin remodeling complex.

To understand the molecular requirements for histone ubiquitylation readout in general and for H2BK120ub readout in particular, we repeated the affinity purification scheme mentioned above with the addition of ubiquitylated H2BK120ub histone and ubiquitylated H2BK120ub mononucleosomes as modified templates (Figure 3.11A, Figure 3.13). The two additional affinity matrices were selected to provide information with regard to the requirement of inter- and trans-nucleosomal contacts as well as additional interaction surfaces (DNA gyres, linker DNA and unmodified histones) for readout of the ubiquitylation mark.

In the histone affinity purification experiment, four proteins were reproducibly enriched (Fig-ure 3.13A, Figure 3.13B). Nuclesomal assembly protein 1-like 1 and 4 (NAP1L1, NAP1L4), nucleolar (upstream) transcription factor (UBTF) and the Treacher-Collins ribosome biogen-esis factor 1 (TCOF1) discriminated ubiquitylated H2B histone from the unmodified control.

In the mononucleosome affinity purification experiment, a group of fourteen proteins were reproducibly enriched (Figure3.13C, Figure3.13D). Four subunits of the integrator complex (INTS 1, INTS3, INTS6 and INTS12) and the RNA polymerase II subunit A (POLR2A) were identified as strong readers. Zinc-finger containing proteins ZMYNDB, ZNF687, struc-tural maintenance of chromosomes 1A and 3 (SMC1A, SMC3) and the E3 protein ligase ring finger 169 (RNF169) also prefered the ubiquitylated mononucleosome matrix to the unmodified control.

In the chromatin affinity purification experiment, thirty-five proteins were reproducibly en-riched (Figure3.13E, Figure3.13F). In agreement with the published H2BK120ub chromatin interactome [83], eleven subunits of the integrator complex (INTS1-INTS10, INTS12) were recruited to the modified chromatin. Three subunits of the RNA polymerase machinery (POLR2B, POLR2C, POLR2E) and several subunits controlling transcription elongation (NELFB, NELFC/D, SUPT5H) were also enriched. DNA excision repair proteins 1 and 4 (ERCC1 and ERCC4) as well as the Zn-finger proteins ZMYNDB, ZNF609 and ZNF687

Figure 3.13: ChaP-MS analysis of H2BK120ub. (A) Representation of enriched interac-tors on an interactome plot from a histone affinity purification experiment. (B) Representation of enriched interactors on a volcano plot from a histone affinity purification experiment. (C) Represen-tation of enriched interactors on an interactome plot from a mononucleosome affinity purification experiment. (D) Representation of enriched interactors on a volcano plot from a mononucleosome affinity purification experiment. (E) Representation of enriched interactors on an interactome plot from a chromatin affinity purification experiment. (F) Representation of enriched interactors on a volcano plot from a chromatin affinity purification experiment.

and the E3 ligase RNF169 preferred the ubiquitylated chromatin array over the unmodified control.

There was no overlap between the factors that were enriched using histone affinity

purifi-of the proteins enriched with the histone matrix werre present in the mononucleosome or the chromatin sets. None of the proteins enriched on the mononucleosome or the chromatin matrices were found within the histone set.

There was overlap between the mononucleosome and chromatin affinity purification experi-ments. Several subunits of the integrator complex, the RNA polymerase and several Zn-finger proteins were found in both experiments. The momonucleosome matrix recruited however only a fraction of the factors enriched on the chromatina arrays.

The nucleosomal arrays provide thus a more complex and larger interaction surface, which translates during the affinity purification into more binding events. The biotiniylated nucle-osomal arrays create efficient affinity matrices, where the nuclear interactors’ need for inter-and trans-nucleosomal contacts is statisfied inter-and where the presence of ubiquitin is readily discriminated.

The interactome of H2BK34ub

Histone and chromatin affinity purification experiments were also used to identify proteins that recognise H2BK34ub.

Using a ubiquitylated histone matrix, the H2BK34ub modification enriched for a number of proteins with seemingly unrelated functions: the phospholipase A2 activating protein (PLAA), the transcriptional regular SUB1, the solute carrier family member 4A1 (SLC4A1), the ADP ribosylation factor like 6 (ARL6), and the Zn-finger RNA binding protein 2 (ZRANB2) (Figure 3.14A, Figure 3.14B).

Using a ubiquitylated chromatin matrix, the H2BK34ub mark reproducibly enriched for six proteins, including the DNA Polymerase E3 (POLE3), the chromatin accessibility complex 1 (CHRAC1), a subunit of the INO80 chromatin remodeling complex and the actin related protein 5 (ACTR5) (Figure 3.14C, Figure 3.14D).

Similarly as for the H2BK120ub modification, there was no overlap between the histone and the chromatin affinity purification datasets collected for H2BK34ub.

None of the interactors enriched on the H2BK120ub histone, mononucleosome or chromatin matrices was identified within the H2BK34ub-specific datasets.

The interactome of H3K18ub and H3K23ub

The chromatin affinity purification mass spectrometry technique was also used to identify the proteins which recognise the H3K18ub, H3K23ub and H3K18/23ub2 modifications.

The proteins purified on the H3K18ub template were more widely distributed than the other two H3 ubiquitylated templates (Figure 3.15A, Figure 3.15B). After applying the statistical

Figure 3.14: ChaP-MS analysis of H2BK34ub. (A) Representation of enriched interactors on an interactome plot from a histone affinity purification experiment. (B) Representation of enriched interactors on a volcano plot from a histone affinity purification experiment. (C) Representation of enriched interactors on an interactome plot from a chromatin affinity purification experiment.

(D) Representation of enriched interactors on a volcano plot from a chromatin affinity purification experiment.

analysis and the enrichment threshold, twenty-eight proteins were found reproducibly en-riched. These included as strongest interactors the DNA methyltransferase 1 (DNMT1), the ubiquitin specific protease 7 (Usp7), the Sex comb on midleg-like 2 (SCML2), the acetyl-coA carboxylase alpha (ACACA) and the ubiquitin specific protease 3 (Usp3). Other enriched interactors included Zn-finger proteins ZBTB1, ZBTB14 and ZBTB44, histone deacetylase components SIN3A and associated subunits SAP30 and SAP 130, transcription regulators Forkhead Box 1 and 2 (FOXK1, FOXK2) as well as MAX dimerization protein MLX and interacting proteins MLXIP and MLXIPL with transcription factor properties.

Using the H3K23ub nucleosomal array, six proteins were enriched by affinity purification (Figure 3.15C, Figure 3.15D). Similarly to the H3K18ub template, DNMT1, Usp7, SCML2 and ACACA were among the enriched factors. In addition, the DNA sensor 5-hydroxymethyl-cytosine binding embryonic stem cell specific (HMCES) and the G2/M phase specific E3 ubiquitin ligase G2E3 also preferred the ubiquitylated template to the unmodified control.

Using the double-modified H3K18/23ub2 nucleosomal array, six proteins were enriched by

Figure 3.15: ChaP-MS analysis of H3 ubiquitylaion. (A) Representation of enriched in-teractors on an interactome plot from a H3K18ub chromatin affinity purification experiment. (B) Representation of enriched interactors on a volcano plot from a H3K18ub chromatin affinity pu-rification experiment. (C) Representation of enriched interactors on an interactome plot from a H3K23ub chromatin affinity purification experiment. (D) Representation of enriched interactors on a volcano plot from a H3K23ub chromatin affinity purification experiment. (E) Representation of enriched interactors on an interactome plot from a H3K18/23ub2 chromatin affinity purification experiment. (F) Representation of enriched interactors on a volcano plot from a H3K18/23ub2 chromatin affinity purification experiment.

periments, this purification enriched for DNMT1, Usp7, SCML2 and ACACA. In addition, it recruited MLX1PL and FOXK2, which were also found on the H3K18ub affinity matrix.

There was overlap between the three H3 ubiquitylated affinity matrices. DNMT1, Usp7,

SCML2 and ACACA were the highest enriched factors in all datasets. There was no over-lap between these three interactomes and the interactomes collected for the H2BK120ub or H2BK34ub chromatin templates.

Complete lists of enriched factors are presented in Table 3.1, Table 3.2 and Table 3.3. For each interactome, the tables list three separate identifications: the two-sample t-test analysis, the application of the H/L enrichment cutoff and the combined use of the H/L enrichment cutoff with the t-test statistical analysis which has been presented in the thesis.

3.2.3 Network analysis of the histone ubiquitylation interactome