A novel chromatin-remodeling complex variant, dcPBAF, is involved in maintaining transcription in differentiated neurons

The Polybromo-associated BAF (BRG1- or BRM-associated factors) (PBAF) chromatin-remodeling complex is essential for transcription in mammalian cells. In this study, we describe a novel variant of the PBAF complex from differentiated neuronal cells, called dcPBAF, that differs from the canonical PBAF existing in proliferating neuroblasts. We describe that in differentiated adult neurons, a specific subunit of PBAF, PHF10, is replaced by a PHF10 isoform that lacks N- and C-terminal domains (called PHF10D). In addition, dcPBAF does not contain the canonical BRD7 subunit. dcPBAF binds promoters of the actively transcribed neuron-specific and housekeeping genes in terminally differentiated neurons of adult mice. Furthermore, in differentiated human neuronal cells, PHF10D-containing dcPBAF maintains a high transcriptional level at several neuron-specific genes.


Supplementary
Figure S1.Schematic representation of PHF10 transcripts.The coding regions are indicated by black and the noncoding regions by gray boxes.Primers for PHF10A measurement depicted by dark-green arrows and primers for PHF10D measurement depicted by yellow arrows (sequences of primers are at the Table S5).Also siRNA-I and siRNA-II for PHF10D knockdown depicted black arrows (sequences of siRNA's are at the Table S4).

Table S1.
The human and murine PHF10 mRNAs and protein isoforms present in the NCBI databases.
The analysis of mouse and human PHF10 transcripts has shown that like other mammals they have the splice variants encoding DPF containing isoform and DPF lacking isoform (Brechalov et al., 2014, Chugunov et. al., 2021).The human PHF10 transcripts were precisely mapped (Brechalov et al., 2014).The Northern blot analysis demonstrates that mouse and human have the same four PHF10 transcripts of the similar length.In Database are annotated mouse transcripts encoding long PHF10 isoforms: Phf10A (NM_024250 -1665 nt, NP_077212.3-497 aa) and Phf10C (NM_001360983 -3992 nt, NP_001347912.1 -376 aa).

Figure S2
. PHF10 isoforms undergo multiple phosphorylation as was confirmed on brain extracts treated with the Lambda-phosphatase (L-PP).PHF10 is comprised of four major bands, corresponding to the described PHF10 isoforms, and several additional bands which appeared due to multiple phosphorylation the PHF10 isoforms (depicted as +Ph).Note that on Figure 1 and after, the PHF10 isoforms are indicated according to the analysis of their electrophoretic mobility performed previously (Tatarskiy et al., 2017).Each PHF10 isoform has its own specific phosphorylation pattern, due to the difference in their structure.PhIC + -nuclear extract prepared with Phosphatase inhibitor cocktail addition.L-PP + -nuclear extract prepared with Lambda phosphatase addition.
. Western Blot of proteins precipitated with antibodies against PHF10 and BRD7 from P1 and P56 extracts.Western blot was developed with antibodies against PHF10 and BRD7 (indicated on the left).The obtained results show that antibodies against PHF10 co-precipitate BRD7 from P1 extract containing PHF10A isoform and PBAF complex.From P56 extract which contains PHF10D (associated with dcPBAF), the antibodies against PHF10 do not co-precipitate BRD7.These results are confirmed in reciprocal precipitation with antibodies against BRD7.

Figure S4
. Western Blot analysis of the PBAF subunits precipitated with antibodies against DPFdomain and against total PHF10 from nuclear extracts of P56 murine brain.Anti-PHF10 antibodies precipitate PHF10D from P56 extract.They also co-precipitate BAF200, BAF180 but not BRD7, suggesting that BAF200 and BAF180 are associated with dcPBAF even in the absence of BRD7.Note that the Input on Supplementary Fig. 5 differs from Input on Supplementary Fig. 6 due to the different amount of extract loaded per lane.

Figure S5 .
Figure S5.The levels of PHF10A and PHF10A isoforms in murine postnatal of brain.The levels of PHF10A and PHF10D isoforms during development were calculated relatively P1 expression.The values were normalized to the actin and B2M housekeeping genes according to MIQE guidelines (Bustin et al., 2009).At least three independent experiments were performed; values are mean ± SD.

Figure S6 .
Figure S6.The distribution of genomic elements in the PHF10 binding sites and in the random sites distributed uniformly along the genome (N=301).

Figure S7 .
Figure S7.The results of ChIPseq showing the peaks of PHF10 from replicates and IgG (PI) at the promoters of mouse neuronal genes Bdnf and MAP2 homologues to human genes shown at Figure 6.

Table S2 .
Proteins co-precipitated by anti-PHF10 antibodies were eluted and identified by MALDI-TOFF.