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A key challenge in synthetic biology is achieving durable amplification of low-level inputs in gene regulation systems. Current RNA-based tools primarily operate post-transcriptionally and often yield limited, transient responses. An underexplored feature of lowly expressed long non-coding RNAs (lncRNAs) is their ability to induce outsized effects on chromatin regulation across large genomic regions. Mechanistic insights from basic research are bringing the field closer to designing lncRNAs for epigenetic engineering. We review foundational studies on ectopic expression to uncover lncRNA-mediated epigenetic mechanisms and state-of-the-art transgenic systems for studying lncRNA-driven epigenetic regulation. We present perspectives on strategies for testing the composability of modular lncRNA elements to build rationally designed systems with programmable chromatin-modifying functions and potential biomedical applications such as gene dosage correction. Deepening mechanistic insights into lncRNA function, combined with the development of lncRNA-based technologies for genome regulation, will pave the way for significant advances in cell state control. 

Regulation of gene silencing in large regions of chromosomes is crucial for development and disease progression, and there has been an increasing interest in using it for new therapeutics. One example of massive gene silencing is X chromosome inactivation (XCI), a process essential for dosage compensation of X-linked genes. During XCI, most genes in the X chromosome are inactivated following the transcription of XIST, an X-linked long noncoding RNA. Recent experiments with transgenes showed that the spread of gene silencing can be induced by XIST transcription in cis, but the spread is restricted in space. The mechanism of controlling the spread remains unclear. In this work, we develop a continuum reaction-diffusion model that elucidates chromosomal inactivation through a bistable system governed by a regulatory network for XIST-mediated gene silencing. We find that the spread of XIST can be tuned by known negative feedback loops regulating its synthesis and degradation, and that the spread of gene silencing is controlled by a wave-pinning mechanism in which both global regulation of silencing complex and local variations of histone modifications can play crucial roles. In addition, we integrate the discrete three-dimensional arrangement of the X chromosome and autosomes into this continuous model. We use a 3D chromosome structure inferred from experimental data and our modeling framework to show the spatiotemporal regulation for spread of gene silencing. Our method enables the investigation for the inactivation dynamics of large regions of chromosomes with varying degrees of the spread of gene silencing. Our model provides mechanistic insights that quantitatively relate gene regulatory networks to tunability and stability of chromosomal inactivation. 

Long noncoding RNAs (lncRNAs), a class of noncoding RNAs exceeding 500 nucleotides and transcribed mostly by RNA polymerase II, regulate gene transcription and chromatin organization. Acting as molecular guides, scaffolds, and structural components, lncRNAs interact with RNAs, DNA, and proteins. Repeat motifs within lncRNAs called k-mers are associated with protein interactions and chromatin complex recruitment. Understanding lncRNA mechanisms is hampered by tissue specificity, redundancy, and multifunctionality, necessitating quantitative investigation. To support the systematic investigation of repeat motifs, we optimized Golden Gate assembly with standardized 4-nucleotide linkers to assemble any four lncRNA elements into 4x k-mers without the need to design compatible overhangs. To extend the length (repeat number) of RNA constructs, a 2x BbsI Golden Gate cloning site is restored at the 3’ end of the assembled 4x k-mer, allowing the addition of more k-mers to generate an 8x k-mer, 12x k-mer, and so on. Please read the Guidelines for suggestions on how to make arrays of intermediate lengths (e.g. 1x, 2x, 3x, 5x, 6x, 7x, etc.). Beginning with k-mer identification, synthetic lncRNAs can be constructed and expressed in vitro or in cells within three weeks, offering an efficient framework to study and harness their regulatory functions.