Capture of AT-rich Chromatin by ELYS Recruits POM121 and NDC1 to Initiate Nuclear Pore Assembly By Rasala et al. Presented by Laurent Palmatier BGGN /02/10

Introduction All eukaryotes have a nuclear envelope (NE) Includes nuclear membranes, and large nuclear pore complexes (NPCs) built by 500 – 1000 proteins The Nucleus Nuclear pore complex (NPC) Nuclear membranes Nuclear membrane and NPC reformation occurs at the end of mitosis This study explores proteins involved in the early NPC reformation

Introduction The study is carried out in vitro using Xenopus eggs and sperm for essential biomolecules (proteins, membranes, chromatin)

Synthesis of an ELYS Antibody ELYS is known to play an early role in NPC formation An antibody stain showed colocalization of ELYS with known nucleoporins (Nups) 5um

Specificity of ELYS localization AL are cytoplasmic membranes with pores Immunoblot studies show ELYS purifies with nuclei but not AL ELYS likely has a binding affinity for chromatin

How does ELYS bind to chromatin? Known to have an AT- hook motif at the C- terminus Carried out mutation experiments to verify this region can bind to chromatin

How does ELYS bind to chromatin? Immunoblot analysis of three samples shows relative binding affinities

Chromatin Binding and NPC formation Inhibition of ELYS and Nup binding to chromatin AT-hook + / AT-hook 2R  A / ΔAT-hook Determining effects on nuclear pore assembly using anti-FG-nucleoporin antibody: Alexa-568-mAb414 10um

Chromatin binding and NPC formation AT-hook + / AT-hook 2R  A / ΔAT-hook Are lower Ran-GTP levels responsible for lack of nuclear pore formation? 10um Excess Ran-GTP AT-hook + acts at the surface of the chromatin, inhibiting binding of endogenous ELYS.

Distamycin A and Chromomycin A 3 Antibiotics Distamycin A and Chromomycin A3 bind to AT and GC rich DNA respectively Distamycin A blocks nuclear pore assembly, but Chromomycin A3 does not 5um

Distamycin A blocks by binding to DNA AL control shows distamycin A inhibits via DNA binding This is because known nucleoporins in the AL are still identified in the presence of distamycin A

Distamycin A inhibits ELYS / chromatin binding Anchored chromatin assay carried out to definitively show distamycin A blocks ELYS/chromatin binding

Nuclear import assays Nuclear import assays with GFP-M9 support previous results 5um

Two Hypotheses: 1) Additional soluble pore subunits recruited to the chromatin/ELYS/ Nup precursor in a membrane-independent manner 2) One or more integral membrane pore protein(s) with associated membrane vesicle or sheet is recruited. Identifying the next step in NPC formation

Testing first hypothesis: 1) Additional soluble pore subunits recruited to the chromatin/ELYS/ Nup precursor in a membrane-independent manner Identifying the next step in NPC formation Tested by anchoring chromatin and immunoblotting for bound proteins Found that recruitment of membrane must follow ELYS and Nup binding to chromatin

POM121 is an integral membrane protein involved in early NPC formation Importin α and β bound to POM121, but were removed by RanQ69L-GTP Nup bonds strongly to POM121 Indicates that POM121 recruitment occurs just after ELYS and Nup binding to the chromatin POM121 is known to function early in NPC formation POM121 fragment with removed FG repeats avoids transport receptor binding, but allows soluble nucleoporins to bind. Pore subunits containing Nup358, Nup214, Nup155, Nup62, and Nup53 showed no affinity for POM121

POM121 recruitment depends on ELYS and Nup localization to the chromatin Distamycin A, and ELYS AT-hook + had been found in this study to block ELYS association to the chromatin Using anti-POM121-AF488, assays for POM121 in four separate experiments show it will not associate without ELYS being bound previously 10um

Recruitment of integral membrane proteins gp210 and NDC1 Immunoblot analysis determined that recruitment of NDC1 needs ELYS bound to chromatin, but recruitment of gp210 does not.

Timeline of NPC formation 1)ELYS binds to the AT-rich chromatin at two binding sites 2)Nup complex associates 3)POM121 and NDC1 are actively recruited 4)The remaining soluble pore subunits are assembled

Summary ELYS binds to chromatin and targets nuclear pore assembly to the surface of chromosomes as nuclei form at the end of mitosis. ELYS is not needed for AL pore formation ELYS C-terminus contains two chromatin binding domains (including the AT hook domain) Excess ELYS AT-hook + fragment blocks recruitment of ELYS and the Nup complex, and therefore NPC assembly (mutant or Δ AT-hook domain does not) Distamycin A inhibits NPC assembly in the same way though Chromomycin A 3 does not Besides ELYS, Nup , and possibly Nup153, the remaining soluble pore subunits require membrane recruitment and fusion to associate with forming NPCs POM121 binds to the Nup complex, showing that its recruitment likely follows ELYS/Nup chromatin Inhibition of ELYS chromatin binding also inhibits the recruitment of POM121 and NDC1

Future Work Transitioning to in vivo experimentation Working with human NEs in case pore formation occurs differently Further characterization of subunit functionalities Studying yeast which lack ELYS Studying specific base pairs which bind ELYS

Capture of AT-rich Chromatin by ELYS Recruits POM121 and NDC1 to Initiate Nuclear Pore Assembly By Rasala et al. Presented by Laurent Palmatier BGGN /02/10