A prelude to the proximity interaction mapping of CXXC5

Abstract

CXXC5 is a member of the zinc-finger CXXC family proteins that interact with unmodified CpG dinucleotides through a conserved ZF-CXXC domain. CXXC5 is involved in the modulation of gene expressions that lead to alterations in diverse cellular events. However, the underlying mechanism of CXXC5-modulated gene expressions remains unclear. Proteins perform their functions in a network of proteins whose identities and amounts change spatiotemporally in response to various stimuli in a lineage-specific manner. Since CXXC5 lacks an intrinsic transcription regulatory function or enzymatic activity but is a DNA binder, CXXC5 by interacting with proteins could act as a scaffold to establish a chromatin state restrictive or permissive for transcription. To initially address this, we utilized the proximity-dependent biotinylation approach. Proximity interaction partners of CXXC5 include DNA and chromatin modifiers, transcription factors/co-regulators, and RNA processors. Of these, CXXC5 through its CXXC domain interacted with EMD, MAZ, and MeCP2. Furthermore, an interplay between CXXC5 and MeCP2 was critical for a subset of CXXC5 target gene expressions. It appears that CXXC5 may act as a nucleation factor in modulating gene expressions. Providing a prelude for CXXC5 actions, our results could also contribute to a better understanding of CXXC5-mediated cellular processes in physiology and pathophysiology.

Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

“…TurboID uses a biotin ligase engineered through yeast display‐based directed evolution of a promiscuous mutant of E. coli biotin ligase BirA* that catalyzes proximity biotin labeling of proteins with higher efficiencies and faster kinetics than BirA*. This allows the investigation of protein interaction networks dynamically (Ayaz et al, 2021 ). Based on our initial analyses that TurboID can be effectively used for dynamic protein interaction profiling of YPEL2 (Figure S4 ), we cloned the Turbo‐HA, 3F‐YPEL2, or 3F‐YPEL2‐Turbo‐HA cDNA into pINDUCER20‐MCS to efficiently dictate the timing and the level of protein synthesis in transiently transfected cells to minimize potential adverse effects of transgene overexpression.…”

“…For YPEL3, we also used transcript variant 1, which has the identical core sequence of transcript variant 2, containing an extended 5′ sequence that encodes an additional 38 amino acids at the amino terminus. In the engineering of all constructs, the first ATG was designed to be embedded into the consensus Kozak sequence (CCATGG) for efficient translation; reverse cloning primers were also designed to contain a polyA signal (TAATAAA) that comprises a stop codon as we described previously (Ayaz et al, 2021 ; Yaşar et al, 2016 ; Yi et al, 2002 ). The generated YPEL cDNAs were then cloned into the pcDNA 3.1(−) vector.…”

“…Protein identification by mass spectrometry . MS analyses were carried out at the Koç University Proteomic Facility (Istanbul, Türkiye) as we described previously (Ayaz et al, 2021 ). The protein‐bound streptavidin beads were washed with 50 mM NH 4 HCO 3 , followed by reduction with 100 mM DTT in 50 mM NH 4 HCO 3 at 56°C for 45 min, and alkylation with 100 mM iodoacetamide at RT in the dark for 30 min.…”

Interactomics of CXXC proteins involved in epigenetic regulation of gene expression

“…Поэтому исследования интерактома CXXC-белков как совокупного спектра взаимодействующих друг с другом индивидуальных белков и белковых комплексов важны с точки зрения получения новых знаний о: а) прямых и непрямых белковых партнёрах, формирующих мультибелковые комплексы; б) модифицирующих белках, таких как протеинкиназы и убиквитиназы; в) "каркасных" белках (от англ. scaffold), которые, в частности, были идентифицированы в качестве белковых партнёров СXXC5 [14].…”

“…Интересно, что спектр мутаций в гене CXXC6 имел значимость для прогноза ответов на лечение ингибиторами контрольных точек иммунитета и общей выживаемости пациентов с разными нозологиями [21]. Как правило, индукция экспрессии большинства генов CXXC в опухолевых тканях коррелирует с развитием высокопролиферативных и инвазивных новообразований [22,23], о чём также свидетельствуют эксперименты по нокдауну генов [14,24,25]. Также было показано, что паттерн экспрессии ассоциирован с неблагоприятным прогнозом онкологического заболевания и метастатическими осложнениями [26,27].…”