Journal: bioRxiv

Article Title: Condensin chromatin association is regulated by SMC head and hinge engagement and phosphorylation

doi: 10.1101/2025.11.17.688780

Figure Lengend Snippet: Expression of inducible Smc2 mutants created by conditional protein exchange. A) Schematic view of budding yeast condensin, illustrating the relative locations of each Smc2 mutation. Engagement of ATP-bound head domains triggers ATP hydrolysis, leading to their disengagement. The K38I and D1112A mutations are predicted to block binding of ATP to their respective pockets. The E113Q mutation is predicted to slow down the rate of ATP hydrolysis, while R58A is predicted to disrupt DNA dependent ATP hydrolysis. The S1085R mutation is predicted to disrupt head-head interactions. B) Within the hinge region of Smc2, we designed two mutations to specifically disrupt either of the two interfaces made with Smc4: hinge interface 1, L665R; hinge interface 2, L567K. Inset panels show additional details for how each single point mutant is predicted to generate a series of steric clashes (red circles) incompatible with hetero-dimerization of the hinge. Key amino acids of Smc2 and Smc4 are labelled in plain and italic text respectively. Figures were generated using MacPyMOL. C) Conditional protein exchange system for smc2 mutations. Under the permissive conditions (2% raffinose, 25°C) functional degron and HA tagged protein is expressed. Under the restrictive conditions (+2% galactose, + doxycycline 37°C), the functional degron protein is degraded and the exogenous HA tagged protein is expressed from a GAL1 promoter. D) Spot test of viability following conditional protein exchange for the different smc2 mutants. E) Condensin complexes generated following expression of different smc2 mutants. Following conditional protein exchange, in vivo condensin complexes were immuno-precipitated and the isolated complexes analyzed by MS/MS as described in the Methods. The signal from several peptides derived from each protein was quantitated by MS/MS and then normalized to the quantity of peptides from the Brn1 subunit. This provides a measure of the relative quantity of each subunit immuno-precipitated by Brn1-V5. All experiments are the average of two independent experiments and the standard deviation of the experiment shown by the error bar.

Article Snippet: Anti-V5 antibody (mouse monoclonal MCA1360, abD Serotec) used at 1:1000 Concentration in Western.

Techniques: Expressing, Mutagenesis, Blocking Assay, Binding Assay, Generated, Functional Assay, Spot Test, In Vivo, Isolation, Tandem Mass Spectroscopy, Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: Condensin chromatin association is regulated by SMC head and hinge engagement and phosphorylation

doi: 10.1101/2025.11.17.688780

Figure Lengend Snippet: Chromatin association of condensin following expression of smc2 enzymatic mutations. Conditional expression depletion strains were arrested in G1 and endogenous Smc2 degraded and the indicated version of exogenous Smc2 expressed. Cells were then released from G1 into restrictive medium containing nocodazole. After 2 h, cells were harvested, and Brn1-V5 levels at the indicated sites analyzed by ChIP-qPCR. The average of at least three experimental repeats (qPCR performed in triplicate in each case) is shown with error bars. Enrichments are grouped according to function,

Article Snippet: Anti-V5 antibody (mouse monoclonal MCA1360, abD Serotec) used at 1:1000 Concentration in Western.

Techniques: Expressing, ChIP-qPCR

Journal: bioRxiv

Article Title: Condensin chromatin association is regulated by SMC head and hinge engagement and phosphorylation

doi: 10.1101/2025.11.17.688780

Figure Lengend Snippet: Effects of phosphorylation defective condensin subunits on chromatin association. Strains expressing the defined phosphorylation mutants of A) Smc4- smc4-10A , B) Brn1- brn1-570 , C) Ycs4 – ycs4-543 D) Ycg1, ycg1-521 were released from G1 into medium containing nocodazole at 37°C. After 2 h, cells were harvested, and Brn1-V5 levels at the indicated sites analyzed by ChIP-qPCR. The average of at least three experimental repeats (qPCR performed in triplicate in each case) is shown with error bars.

Article Snippet: Anti-V5 antibody (mouse monoclonal MCA1360, abD Serotec) used at 1:1000 Concentration in Western.

Techniques: Phospho-proteomics, Expressing, ChIP-qPCR

Journal: bioRxiv

Article Title: Condensin chromatin association is regulated by SMC head and hinge engagement and phosphorylation

doi: 10.1101/2025.11.17.688780

Figure Lengend Snippet: Requirement for Ipl1 and Cdc5 for condensin chromatin association and complex stability. Either A) ipl1-321 , B) cdc5-10 C) cdc5-99 cells were released from G1 into medium containing nocodazole at 37°C. After 2 h, cells were harvested, and Brn1-V5 levels at the indicated sites analyzed by ChIP-qPCR. The average of at least three experimental repeats (qPCR performed in triplicate in each case) is shown with error bars.

Article Snippet: Anti-V5 antibody (mouse monoclonal MCA1360, abD Serotec) used at 1:1000 Concentration in Western.

Techniques: ChIP-qPCR

Journal: NPJ Genomic Medicine

Article Title: Disruption of SPECC1L translation initiation by intragenic deletion: novel pathogenic mechanism in Teebi-hypertelorism syndrome

doi: 10.1038/s41525-025-00513-4

Figure Lengend Snippet: a Linearized representation of the SPECC1L open reading frame constructs highlighting the CMV promoter region (purple), the non-coding (unfilled) and coding exons (filled, amber), V5 tag (pink) and the polyA termination signal (blue), canonical and predicted alternative start codons denoted by a green diamond and red diamonds correspondingly. b Immunoblotting of the SPECC1L wild-type (WT, lanes 2–4, approximately 160 kDa) and mutant (exon 3 deletion, ex3del, lanes 5-7, approximately 150 kDa, highlighted by a red diamond) samples; lane 1: protein ladder (L) with reference band sizes indicated. ERK1/2 was used as a loading control. c Multi-species alignment of the first 150 amino acids of the disordered region of the SPECC1L reference protein, highlighting potential in-frame alternative start codons (conserved methionines at M62, M91, and M148 positions of human SPECC1L (UniProtKB # Q69YQ0 ), in red); the region corresponding to the patients’ deletion of the first 51 amino acids is outlined by a red box.

Article Snippet: The membrane was then blocked in a 5% milk-TBST for 1 h, then sectioned and incubated in 5% milk-TBST with primary antibodies against V5 (Bio-Rad, #MCA1360, 1:1000) and ERK1/2 (loading control, Cell Signalling Technology, #9102, 1:1000) respectively for 2 h at RT.

Techniques: Construct, Western Blot, Mutagenesis, Control